Comprehensive genome sequence analysis of Ralstonia solanacearum gd-2, a phylotype I sequevar 15 strain collected from a tobacco bacterial phytopathogen.

Introduction: Plant bacterial wilt is an important worldwide disease caused by Ralstonia solanacearum which is a complex of species. Methods: In this study, we identified and sequenced the genome of R. solanacearum strain gd-2 isolated from tobacco. Results: Strain gd-2 was identified as R. solanacearum species complex (RSSC) phylotype I sequevar 15 and exhibited strong pathogenicity to tobacco. The genome size of gd-2 was 5.93 Mb, including the chromosomes (3.83 Mb) and the megaplasmid (2.10 Mb). Gene prediction results showed that 3,434 and 1,640 genes were identified in the chromosomes and plasmids, respectively. Comparative genomic analysis showed that gd-2 exhibited high conservation with ten highly similar strain genomes and the differences between gd-2 and other genomes were mainly located at positions GI12-GI14. 72 type III effectors (T3Es) were identified and RipAZ2 was a T3E specific to gd-2 compared with other eight sequenced strain. Discussion: Our study provides a new basis and evidence for studying the pathogenic mechanism of R. solanacearum.

Genome-wide identification of the TIFY gene family in tobacco and expression analysis in response to Ralstonia solanacearum infection.

The TIFY gene family plays an essential role in plant development and abiotic and biotic stress responses. In this study, genome-wide identification of TIFY members in tobacco and their expression pattern analysis in response to Ralstonia solanacearum infection were performed. A total of 33 TIFY genes were identified, including the TIFY, PPD, ZIM&ZML and JAZ subfamilies. Promoter analysis results indicated that a quantity of light-response, drought-response, SA-response and JA-response cis-elements exist in promoter regions. The TIFY gene family exhibited expansion and possessed gene redundancy resulting from tobacco ploidy change. In addition, most NtTIFYs equivalently expressed in roots, stems and leaves, while NtTIFY1, NtTIFY4, NtTIFY18 and NtTIFY30 preferentially expressed in roots. The JAZ III clade showed significant expression changes after inoculation with R. solanacearum, and the expression of NtTIFY7 in resistant varieties, compared with susceptible varieties, was more stably induced. Furthermore, NtTIFY7-silenced plants, compared with the control plants, were more susceptible to bacterial wilt. These results lay a foundation for exploring the evolutionary history of TIFY gene family and revealing gene function of NtTIFYs in tobacco bacterial wilt resistance.

Integrated transmission electron microscopy and proteomic analyses reveal the cytoarchitectural response to cucumber mosaic virus infection in tobacco.

Cucumber mosaic virus (CMV) causes huge economic losses to agriculture every year; thus, understanding the mechanism of plant resistance to CMV is imperative. In this study, an integrated analysis of transmission electron microscopy (TEM) observations and proteomic results was used to identify cytoarchitectural differences in Nicotiana tabacum cv. NC82 (susceptible) and cv. Taiyan 8 (T.T.8; resistant) following infection with CMV. The TEM observations showed that the structure of the chloroplasts and mitochondria was severely damaged at the late stage of infection in NC82. Moreover, the chloroplast stroma and mitochondrial cristae were reduced and disaggregated. However, in T.T.8, organelle structure remained largely intact Selective autophagy predominated in T.T.8, whereas non-selective autophagy dominated in NC82, resembling cellular disorder. Proteomic analysis of T.T.8 revealed differentially expressed proteins (DEPs) mostly associated with photosynthesis, respiration, reactive oxygen species (ROS) scavenging, and cellular autophagy. Biochemical analyses revealed that ROS-related catalase, autophagy-related disulfide isomerase, and jasmonic acid and antioxidant secondary metabolite synthesis-related 4-coumarate:CoA ligase (Nt4CL) exhibited different trends and significant differences in expression in the two cultivars after CMV inoculation. Furthermore, mutant phenotyping verified that reduced Nt4CL expression impaired resistance in T.T.8. The identified DEPs are crucial for maintaining intracellular homeostatic balance and likely contribute to the mechanism of CMV resistance in tobacco. These findings increase our understanding of plant cytological mechanisms conferring resistance to CMV infection.

Deciphering the roles of tobacco MYB transcription factors in environmental stress tolerance.

The MYB members play important roles in development, metabolism, and stress tolerance in plants. In the current study, a total of 246 tobacco R2R3-MYB transcription factors were identified and systemically analyzed from the latest genome annotation. The newly identified tobacco members were divided into 33 subgroups together with the Arabidopsis members. Furthermore, 44 NtMYB gene pairs were identified to arise from duplication events, which might lead to the expansion of tobacco MYB genes. The expression patterns were revealed by transcriptomic analysis. Notably, the results from phylogenetic analysis, synthetic analysis, and expression analysis were integrated to predict the potential functions of these members. Particularly, NtMYB102 was found to act as the homolog of AtMYB70 and significantly induced by drought and salt treatments. The further assays revealed that NtMYB102 had transcriptional activities, and the overexpression of the encoding gene enhanced the drought and salt stress tolerance in transgenic tobacco. The results of this study may be relevant for future functional analyses of the MYB genes in tobacco.

Systematic Analysis of Tobacco CrRLK1L Family Genes and Functional Identification of NtCrRLK1L47 in Environmental Stresses.

The Catharanthus roseus RLK1-like (CrRLK1L) family is involved in the regulation of plant reproduction, growth and development, cell wall integrity sensing, as well as responses to both biotic and abiotic stress conditions. Extraordinary progress has been made in elucidating the CrRLK1L family receptor kinases-mediated signaling pathway, while limited research addressed the functions of CrRLK1L proteins in tobacco. In this study, we identified and analyzed 48 NtCrRLK1L members from the tobacco genome. The newly identified NtCrRLK1L members were divided into seven groups together with the Arabidopsis CrRLK1L members. The syntenic analysis revealed that four pairs of NtCrRLK1L genes were predicted to have arisen from segmental duplication events. Expression profiling showed that the NtCrRLK1L genes were expressed in various tissues, and most NtCrRLK1L genes were induced by salt and drought stress conditions. Notably, NtCrRLK1L47 was upregulated under drought and salinity stresses, and the NtCrRLK1L47-GFP fusion protein was located in the cell membrane. Furthermore, overexpression of the NtCrRLK1L47 gene enhanced the salt tolerance in tobacco seedlings.

Identification of QTLs Associated With Agronomic Traits in Tobacco via a Biparental Population and an Eight-Way MAGIC Population.

Agronomic traits such as plant height (PH), leaf number (LN), leaf length (LL), and leaf width (LW), which are closely related to yield and quality, are important in tobacco (Nicotiana tabacum L.). To identify quantitative trait loci (QTLs) associated with agronomic traits in tobacco, 209 recombinant inbred lines (RILs) and 537 multiparent advanced generation intercross (MAGIC) lines were developed. The biparental RIL and MAGIC lines were genotyped using a 430 K single-nucleotide polymorphism (SNP) chip assay, and their agronomic traits were repeatedly evaluated under different conditions. A total of 43 QTLs associated with agronomic traits were identified through a combination of linkage mapping (LM) and association mapping (AM) methods. Among these 43 QTLs, three major QTLs, namely qPH13-3, qPH17-1, and qLW20-1, were repeatedly identified by the use of various genetically diverse populations across different environments. The candidate genes for these major QTLs were subsequently predicted. Validation and utilization of the major QTL qLW20-1 for the improvement of LW in tobacco were investigated. These results could be applied to molecular marker-assisted selection (MAS) for breeding important agronomic traits in tobacco.

NtNAC053, A Novel NAC Transcription Factor, Confers Drought and Salt Tolerances in Tobacco.

The NAC (NAM, ATAF1/2, and CUC2) family acts as one of the largest families of the transcription factor in the plant kingdom and was revealed to function as the important regulators in various environmental stresses. However, a few studies were reported about the biofunctions of the NAC transcription factor in tobacco. In the current study, we characterized a novel NAC transcription factor encoding the gene NtNAC053 in tobacco, which was significantly up-regulated when exposed to salt and drought treatments. The results of cis-acting elements analysis suggested that the promoter region of NtNAC053 gene possesses a number of stress-responsive elements, and this gene could be induced by exogenous abscisic acid (ABA) treatment. Moreover, the NtNAC053-GFP fusion protein was localized in the cell nucleus and possessed a transactivation domain in its C-terminal, implying that NtNAC053 may undertake as a transcriptional activator in tobacco. Notably, the overexpression of NtNAC053 in tobacco resulted in hypersensitivity to ABA treatment. Furthermore, these overexpression lines showed significantly enhanced tolerances to drought and salt stresses. Under salt and drought stresses, these overexpression lines possessed higher superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities. Interestingly, the expressions of putative stress-related genes, including NtCOR15A, NtRAB18, NtDREB1A, NtERF5, NtKAT2, and NtERD11, were up-regulated in these overexpression lines when subjected to salt and drought stresses. The clues provided in our study suggested that the NtNAC053 gene encodes a novel NAC transcription factor and could confer the drought and salt stress tolerances by inspiring the downstream stress-responsive genes and antioxidant system in tobacco.

Development of a MAGIC population and high-resolution quantitative trait mapping for nicotine content in tobacco.

Multiparent Advanced Generation Inter-Cross (MAGIC) population is an ideal genetic and breeding material for quantitative trait locus (QTL) mapping and molecular breeding. In this study, a MAGIC population derived from eight tobacco parents was developed. Eight parents and 560 homozygous lines were genotyped by a 430K single-nucleotide polymorphism (SNP) chip assay and phenotyped for nicotine content under different conditions. Four QTLs associated with nicotine content were detected by genome-wide association mapping (GWAS), and one major QTL, named qNIC7-1, was mapped repeatedly under different conditions. Furthermore, by combining forward mapping, bioinformatics analysis and gene editing, we identified an ethylene response factor (ERF) transcription factor as a candidate gene underlying the major QTL qNIC7-1 for nicotine content in tobacco. A presence/absence variation (PAV) at qNIC7-1 confers changes in nicotine content. Overall, the large size of this MAGIC population, diverse genetic composition, balanced parental contributions and high levels of recombination all contribute to its value as a genetic and breeding resource. The application of the tobacco MAGIC population for QTL mapping and detecting rare allelic variation was demonstrated using nicotine content as a proof of principle.

Seed-Specific Expression of the Arabidopsis AtMAP18 Gene Increases both Lysine and Total Protein Content in Maize.

Lysine is the most limiting essential amino acid for animal nutrition in maize grains. Expression of naturally lysine-rich protein genes can increase the lysine and protein contents in maize seeds. AtMAP18 from Arabidopsis thaliana encoding a microtubule-associated protein with high-lysine content was introduced into the maize genome with the seed-specific promoter F128. The protein and lysine contents of different transgenic offspring were increased prominently in the six continuous generations investigated. Expression of AtMAP18 increased both zein and non-zein protein in the transgenic endosperm. Compared with the wild type, more protein bodies were observed in the endosperm of transgenic maize. These results implied that, as a cytoskeleton binding protein, AtMAP18 facilitated the formation of protein bodies, which led to accumulation of both zein and non-zein proteins in the transgenic maize grains. Furthermore, F1 hybrid lines with high lysine, high protein and excellent agronomic traits were obtained by hybridizing T6 transgenic offspring with other wild type inbred lines. This article provides evidence supporting the use of cytoskeleton-associated proteins to improve the nutritional value of maize.

Guanine nucleotide exchange factor 2 for Rab5 proteins coordinated with GLUP6/GEF regulates the intracellular transport of the proglutelin from the Golgi apparatus to the protein storage vacuole in rice endosperm.

Rice glutelin polypeptides are initially synthesized on the endoplasmic reticulum (ER) membrane as a proglutelin, which are then transported to the protein storage vacuole (PSV) via the Golgi apparatus. Rab5 and its cognate activator guanine nucleotide exchange factor (GEF) are essential for the intracellular transport of proglutelin from the Golgi apparatus to the PSV. Results from previous studies showed that the double recessive type of glup4/rab5a and glup6/gef mutant accumulated much higher amounts of proglutelin than either parent line. The present study demonstrates that the double recessive type of glup4/rab5a and glup6/gef mutant showed not only elevated proglutelin levels and much larger paramural bodies but also reduced the number and size of PSVs, indicating a synergistic mutation effect. These observations led us to the hypothesis that other isoforms of Rab5 and GEF also participate in the intracellular transport of rice glutelin. A database search identified a novel guanine nucleotide exchange factor, Rab5-GEF2. Like GLUP6/GEF, Rab5-GEF2 was capable of activating Rab5a and two other Rab5 isoforms in in vitro GTP/GDP exchange assays. GEF proteins consist of the helical bundle (HB) domain at the N-terminus, Vps9 domain, and a C-terminal region. By the deletion analysis of GEFs, the HB domain was found essential for the activation of Rab5 proteins.

A guanine nucleotide exchange factor for Rab5 proteins is essential for intracellular transport of the proglutelin from the Golgi apparatus to the protein storage vacuole in rice endosperm.

Rice (Oryza sativa) glutelins are synthesized on the endoplasmic reticulum as a precursor, which are then transported via the Golgi to protein storage vacuoles (PSVs), where they are proteolytically processed into acidic and basic subunits. The glutelin precursor mutant6 (glup6) accumulates abnormally large amounts of proglutelin. Map-base cloning studies showed that glup6 was a loss-of-function mutant of guanine nucleotide exchange factor (GEF), which activates Rab GTPase, a key regulator of membrane trafficking. Immunofluorescence studies showed that the transport of proglutelins and α-globulins to PSV was disrupted in glup6 endosperm. Secreted granules of glutelin and α-globulin were readily observed in young glup6 endosperm, followed by the formation of large dilated paramural bodies (PMBs) containing both proteins as the endosperm matures. The PMBs also contained membrane biomarkers for the Golgi and prevacuolar compartment as well as the cell wall component, ß-glucan. Direct evidence was gathered showing that GLUP6/GEF activated in vitro GLUP4/Rab5 as well as several Arabidopsis (Arabidopsis thaliana) Rab5 isoforms to the GTP-bound form. Therefore, loss-of-function mutations in GEF or Rab5 disrupt the normal transport of proglutelin from the Golgi to PSVs, resulting in the initial extracellular secretion of these proteins followed, in turn, by the formation of PMBs. Overall, our results indicate that GLUP6/GEF is the activator of Rab5 GTPase and that the cycling of GTP- and GDP-bound forms of this regulatory protein is essential for the intracellular transport of proglutelin and α-globulin from the Golgi to PSVs and in the maintenance of the general structural organization of the endomembrane system in rice seeds.

The small GTPase Rab5a is essential for intracellular transport of proglutelin from the Golgi apparatus to the protein storage vacuole and endosomal membrane organization in developing rice endosperm.

Rice (Oryza sativa) glutelins are synthesized on the endoplasmic reticulum as larger precursors, which are then transported via the Golgi to the protein storage vacuole (PSV), where they are processed into acidic and basic subunits. Three independent glutelin precursor mutant4 (glup4) rice lines, which accumulated elevated levels of proglutelin over the wild type, were identified as loss-of-function mutants of Rab5a, the small GTPase involved in vesicular membrane transport. In addition to the plasma membrane, Rab5a colocalizes with glutelins on the Golgi apparatus, Golgi-derived dense vesicles, and the PSV, suggesting that Rab5a participates in the transport of the proglutelin from the Golgi to the PSV. This spatial distribution pattern was dramatically altered in the glup4 mutants. Numerous smaller protein bodies containing glutelin and α-globulin were evident, and the proteins were secreted extracellularly. Moreover, all three independent glup4 allelic lines displayed the novel appearance of a large dilated, structurally complex paramural body containing proglutelins, α-globulins, membrane biomarkers for the Golgi apparatus, prevacuolar compartment, PSV, and the endoplasmic reticulum luminal chaperones BiP and protein disulfide isomerase as well as ß-glucan. These results indicate that the formation of the paramural bodies in glup4 endosperm was due to a significant disruption of endocytosis and membrane vesicular transport by Rab5a loss of function. Overall, Rab5a is required not only for the intracellular transport of proglutelins from the Golgi to the PSV in rice endosperm but also in the maintenance of the general structural organization of the endomembrane system in developing rice seeds.