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Bacterial wilt, caused by the Ralstonia solanacearum species complex, is one of the most damaging bacterial diseases in tobacco and other Solanaceae crops. In this study, we conducted an analysis and comparison of transcriptome landscape changes in seedling roots of three tobacco BC4F5 lines, C244, C010, and C035, with different resistance to bacterial wilt at 3, 9, 24, and 48 h after R. solanacearum infection. A number of biological processes were highlighted for their differential enrichment between C244, C010, and C035, especially those associated with cell wall development, protein quality control, and stress response. Hence, we performed a genome-wide identification of seven cell wall development-related gene families and six heat shock protein (Hsp) families and proposed that genes induced by R. solanacearum and showing distinct expression patterns in C244, C010, and C035 could serve as a potential gene resource for enhancing bacterial wilt resistance. Additionally, a comparative transcriptome analysis of R. solanacearum-inoculated root samples from C244 and C035, as well as C010 and C035, resulted in the identification of a further 33 candidate genes, of which Nitab4.5_0007488g0040, a member of the pathogenesis-related protein 1 (PR-1) family, was found to positively regulate bacterial wilt resistance, supported by real-time quantitative PCR (qRT-PCR) and virus-induced gene silencing (VIGS) assays. Our results contribute to a better understanding of molecular mechanisms underlying bacterial wilt resistance and provide novel alternative genes for resistance improvement. [ABSTRACT FROM AUTHOR]

Background: Tobacco mosaic virus (TMV) is a widely distributed viral disease that threatens many vegetables and horticultural species. Using the resistance gene N which induces a hypersensitivity reaction, is a common strategy for controlling this disease in tobacco (Nicotiana tabacum L.). However, N gene-mediated resistance has its limitations, consequently, identifying resistance genes from resistant germplasms and developing resistant cultivars is an ideal strategy for controlling the damage caused by TMV. Results: Here, we identified highly TMV-resistant tobacco germplasm, JT88, with markedly reduced viral accumulation following TMV infection. We mapped and cloned two tobamovirus multiplication protein 2A (TOM2A) homeologs responsible for TMV replication using an F2 population derived from a cross between the TMV-susceptible cultivar K326 and the TMV-resistant cultivar JT88. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated loss-of-function mutations of two NtTOM2A homeologs almost completely suppressed TMV replication; however, the single gene mutants showed symptoms similar to those of the wild type. Moreover, NtTOM2A natural mutations were rarely detected in 577 tobacco germplasms, and CRISPR/Cas9-mediated variation of NtTOM2A led to shortened plant height, these results indicating that the natural variations in NtTOM2A were rarely applied in tobacco breeding and the NtTOM2A maybe has an impact on growth and development. Conclusions: The two NtTOM2A homeologs are functionally redundant and negatively regulate TMV resistance. These results deepen our understanding of the molecular mechanisms underlying TMV resistance in tobacco and provide important information for the potential application of NtTOM2A in TMV resistance breeding. [ABSTRACT FROM AUTHOR]

Leaf morphology and plant height are two agronomic traits closely related to tobacco (Nicotiana tabacum L.) yield and quality. The study of leaf morphology and plant stature mutants will greatly contribute to the fields of plant architecture breeding and developmental biology. Here, we report the characterization of a spiral leaf and dwarf (sld) mutant identified from an ethylmethane sulfonate (EMS)-induced common tobacco population. The sld mutant displayed the phenotype of wrinkled, spiral, and miniature leaves, with the growth point as the central axis and plant dwarfing with shortened internodes. The inheritance pattern of the sld mutant phenotype was manipulated by a recessive nuclear monogene, which was linked to six tobacco simple sequence repeat (SSR) markers from linkage group 5 via gene mapping. Utilizing an F2 population, the sld mutant gene the sld mutant gene was located between the co-segregated markers PT51778, PT54913, and the marker PT61414, with an equal genetic distance of 0.16 cM. Taking advantage of a BC1F1 population, the markers PT51778, PT54913, the sld gene, and the marker PT61414 demonstrated co-segregation, located between the markers PT40040 and PT60933, respectively, with a genetic distance of 1.37 cM and 6.32 cM, respectively. These findings will be helpful in cloning the sld gene and in the further characterization of the regulatory genes controlling the spiral and dwarfing phenotypes in tobacco. [ABSTRACT FROM AUTHOR]

Tobacco black shank caused by Phytophthora nicotianae is a serious disease in tobacco cultivation. We found that naringenin is a key factor that causes different sensitivity to P. nicotianae between resistant and susceptible tobacco. The level of basal flavonoids in resistant tobacco was distinct from that in susceptible tobacco. Of all flavonoids with different content, naringenin showed the best antimicrobial activity against mycelial growth and sporangia production of P. nicotianae in vitro. However, naringenin showed very low or no antimicrobial activity to other plant pathogens. We found that naringenin induced not only the accumulation of reactive oxygen species, but also the expression of salicylic acid biosynthesis‐related genes. Naringenin induced the expression of the basal pathogen resistance gene PR1 and the SAR8.2 gene that contributes to plant resistance to P. nicotianae. We then interfered with the expression of the chalcone synthase (NtCHS) gene, the key gene of the naringenin synthesis pathway, to inhibit naringenin biosynthesis. NtCHS‐RNAi rendered tobacco highly sensitive to P. nicotianae, but there was no change in susceptibility to another plant pathogen, Ralstonia solanacearum. Finally, exogenous application of naringenin on susceptible tobacco enhanced resistance to P. nicotianae and naringenin was very stable in this environment. Our findings revealed that naringenin plays a core role in the defence against P. nicotianae and expanded the possibilities for the application of plant secondary metabolites in the control of P. nicotianae. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Ralstonia solanacearum species complex (RSSC) is a diverse group of plant pathogens that attack a wide range of hosts and cause devastating losses worldwide. In this study, we conducted a comprehensive analysis of 131 RSSC strains to detect their genetic diversity, pathogenicity, and evolution dynamics. Average nucleotide identity analysis was performed to explore the genomic relatedness among these strains, and finally obtained an open pangenome with 32,961 gene families. To better understand the diverse evolution and pathogenicity, we also conducted a series of analyses of virulence factors (VFs) and horizontal gene transfer (HGT) in the pangenome and at the single genome level. The distribution of VFs and mobile genetic elements (MGEs) showed significant differences among different groups and strains, which were consistent with the new nomenclatures of the RSSC with three distinct species. Further functional analysis showed that most HGT events conferred from Burkholderiales and played a great role in shaping the genomic plasticity and genetic diversity of RSSC genomes. Our work provides insights into the genetic polymorphism, evolution dynamics, and pathogenetic variety of RSSC and provides strong supports for the new taxonomic classification, as well as abundant resources for studying host specificity and pathogen emergence. [ABSTRACT FROM AUTHOR]

Tobacco mosaic virus (TMV) caused serious loss in yield and quality of tobacco every year. It is a long-term goal to improve the tobacco resistance against TMV by tobacco breeding. N gene was the firstly reported TMV-resistant gene, which showed resistance against all Tobamoviruses except the Ob stain and belonged to the toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat class of plant resistance (R) genes. At present, N gene had already been widely used in tobacco conventional breeding, but there is rare available molecular maker used in marker-assisted selection of TMV resistance. In this study, we designed a pair of primers that specific amplify N gene fragment based on the sequence of N gene intron III, named N-marker. Then, we identified TMV resistance by two selecting methods, PCR with N-marker and inoculated with the TMV-C strain. Results from the two method showed that (1) 13 varieties among 67 tobacco varieties displayed hypersensitive reaction when inoculated with the TMV-C strain, also contained N gene fragments screened by PCR with N-marker; (2) 105 strains of 200 BC1 strains showed resistance against TMV when inoculated with TMV-C strain, meanwhile, 103 of the 105 strains contained N gene fragment verified by PCR with N-marker. Therefore, the N-marker is reliable for high throughput screening of germplasm resources and tobacco breeding materials in selection of N-mediated TMV resistance. Our study not only developed a molecular marker for tobacco breeding, but also identified new germplasm resources that are resistant to TMV. [ABSTRACT FROM AUTHOR]

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 ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Xiao, Li, Yang, Liu, Ren, Cheng, Liu, Jiang, Wen, Wu, Cheng, Yu and Geng.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yuan, Sun, Yu, Jiang, Jiang, Liu, Wen, Si, Wu, Meng, Cheng, Yang and Wang.)

Black shank, caused by the oomycetes Phytophthora nicotianae, is destructive to tobacco, and this pathogen is highly pathogenic to many solanaceous crops. P. nicotianae is well adapted to high temperatures; therefore, research on this pathogen is gaining importance in agriculture worldwide because of global warming. P. nicotianae-resistant varieties of tobacco plants are commonly screened by inoculation with oat grains colonized by P. nicotianae and monitoring for the disease symptoms. However, it is difficult to quantify the inoculation intensity since accurate inoculation is crucial in this case. This study aimed to develop an efficient and reliable method for evaluating the resistance of tobacco to infection with P. nicotianae. This method has been successfully used to identify resistant varieties, and the inoculation efficiency was confirmed by real-time PCR. The resistance evaluation method presented in this study is efficient and practical for precision breeding, as well as molecular mechanism research.