Uncovering the transcriptional responses of tobacco (Nicotiana tabacum L.) roots to Ralstonia solanacearum infection: a comparative study of resistant and susceptible cultivars.

BACKGROUND: Tobacco bacterial wilt (TBW) caused by Ralstonia solanacearum is the most serious soil-borne disease of tobacco that significantly reduces crop yield. However, the limited availability of resistance in tobacco hinders breeding efforts for this disease. RESULTS: In this study, we conducted hydroponic experiments for the root expression profiles of D101 (resistant) and Honghuadajinyuan (susceptible) cultivars in response to BW infection at 0 h, 6 h, 1 d, 3 d, and 7d to explore the defense mechanisms of BW resistance in tobacco. As a result, 20,711 and 16,663 (total: 23,568) differentially expressed genes (DEGs) were identified in the resistant and susceptible cultivars, respectively. In brief, at 6 h, 1 d, 3 d, and 7 d, the resistant cultivar showed upregulation of 1553, 1124, 2583, and 7512 genes, while the susceptible cultivar showed downregulation of 1213, 1295, 813, and 7735 genes. Similarly, across these time points, the resistant cultivar had downregulation of 1034, 749, 1686, and 11,086 genes, whereas the susceptible cultivar had upregulation of 1953, 1790, 2334, and 6380 genes. The resistant cultivar had more up-regulated genes at 3 d and 7 d than the susceptible cultivar, indicating that the resistant cultivar has a more robust defense response against the pathogen. The GO and KEGG enrichment analysis showed that these genes are involved in responses to oxidative stress, plant-pathogen interactions, cell walls, glutathione and phenylalanine metabolism, and plant hormone signal transduction. Among the DEGs, 239 potential candidate genes were detected, including 49 phenylpropane/flavonoids pathway-associated, 45 glutathione metabolic pathway-associated, 47 WRKY, 48 ERFs, eight ARFs, 26 pathogenesis-related genes (PRs), and 14 short-chain dehydrogenase/reductase genes. In addition, two highly expressed novel genes (MSTRG.61386-R1B-17 and MSTRG.61568) encoding nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins were identified in both cultivars at 7 d. CONCLUSIONS: This study revealed significant enrichment of DEGs in GO and KEGG terms linked to glutathione, flavonoids, and phenylpropane pathways, indicating the potential role of glutathione and flavonoids in early BW resistance in tobacco roots. These findings offer fundamental insight for further exploration of the genetic architecture and molecular mechanisms of BW resistance in tobacco and solanaceous plants at the molecular level.

Genome-wide identification and development of InDel markers in tobacco (Nicotiana tabacum L.) using RAD-seq.

Insertions and deletions (InDels) can be used as molecular markers in genetic studies and marker-assisted selection breeding. However, genetic improvement in tobacco has been hindered by limited genetic diversity information and relatedness within available germplasm. A Chinese tobacco variety, Yueyan-98, was resequenced using restriction-site associated DNA (RAD-seq) approach to develop InDel markers. In total, 32,884 InDel loci were detected between Yueyan-98 and the K326 reference sequence [18,598 (56.55%) deletions and 14,288 (43.45%) insertions], ranging from 1 to 62 bp in length. Of the 6,733 InDels (> 4 bp) that were suitable for polyacrylamide gel electrophoresis, 150 were randomly selected. These 150 InDels were unevenly distributed on 23 chromosomes, and the highest numbers of InDels were observed on chromosomes Nt05, Nt13, and Nt23. The average density of adjacent InDels was 19.36 Mb. Thirty-seven InDels were located in genic regions. Polymerase chain reaction (PCR)-based markers were developed to validate polymorphism; 113 (79.80%) of the 150 InDel markers showed polymorphism and were further used for genetic diversity analysis of 50 tobacco accessions (13 from China, 1 from Mexico, and 36 from the USA). The average expected heterozygosity (He) and polymorphism information content (PIC) values were 0.28 ± 0.16 and 0.38 ± 0.10, respectively. The average Shannon diversity index (I) was 0.34 ± 0.18, with genetic diversity ranging from 0.13-0.57. The 50 accessions were classified into two groups with a genetic similarity coefficient of 0.68. Principal coordinate analysis (PCoA) and population structure analysis showed similar results and divided the population into two groups unrelated to their geographical origins. AMOVA showed 4% variance among the population and the remaining 96% within the population, suggesting low genetic differentiation between two subpopulations. Furthermore, 10 InDels (19 alleles) were significantly identified for tobacco plant height using GLM+Q model at P < 0.005. Among these, three markers (Nt-I-26, Nt-I-41, and Nt-I-44) were detected in at least two environments, with phenotypic variance explained (PVE) ranging from 14.03 to 32.68%. The polymorphic InDel markers developed can be used for hybrid identification, genetic diversity, genetic linkage map construction, gene mapping, and MAS breeding programs of tobacco. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01187-3.

Comparative Transcriptome Profiling Reveals Defense-Related Genes Against Ralstonia solanacearum Infection in Tobacco.

Bacterial wilt (BW) caused by Ralstonia solanacearum (R. solanacearum), is a vascular disease affecting diverse solanaceous crops and causing tremendous damage to crop production. However, our knowledge of the mechanism underlying its resistance or susceptibility is very limited. In this study, we characterized the physiological differences and compared the defense-related transcriptomes of two tobacco varieties, 4411-3 (highly resistant, HR) and K326 (moderately resistant, MR), after R. solanacearum infection at 0, 10, and 17 days after inoculation (dpi). A total of 3967 differentially expressed genes (DEGs) were identified between the HR and MR genotypes under mock condition at three time points, including1395 up-regulated genes in the HR genotype and 2640 up-regulated genes in the MR genotype. Also, 6,233 and 21,541 DEGs were induced in the HR and MR genotypes after R. solanacearum infection, respectively. Furthermore, GO and KEGG analyses revealed that DEGs in the HR genotype were related to the cell wall, starch and sucrose metabolism, glutathione metabolism, ABC transporters, endocytosis, glycerolipid metabolism, and glycerophospholipid metabolism. The defense-related genes generally showed genotype-specific regulation and expression differences after R. solanacearum infection. In addition, genes related to auxin and ABA were dramatically up-regulated in the HR genotype. The contents of auxin and ABA in the MR genotype were significantly higher than those in the HR genotype after R. solanacearum infection, providing insight into the defense mechanisms of tobacco. Altogether, these results clarify the physiological and transcriptional regulation of R. solanacearum resistance infection in tobacco, and improve our understanding of the molecular mechanism underlying the plant-pathogen interaction.

Identification of Novel Quantitative Trait Nucleotides and Candidate Genes for Bacterial Wilt Resistance in Tobacco (Nicotiana tabacum L.) Using Genotyping-by-Sequencing and Multi-Locus Genome-Wide Association Studies.

Tobacco bacterial wilt (TBW) is a devastating soil-borne disease threatening the yield and quality of tobacco. However, its genetic foundations are not fully understood. In this study, we identified 126,602 high-quality single-nucleotide polymorphisms (SNPs) in 94 tobacco accessions using genotyping-by-sequencing (GBS) and a 94.56 KB linkage disequilibrium (LD) decay rate for candidate gene selection. The population structure analysis revealed two subpopulations with 37 and 57 tobacco accessions. Four multi-locus genome-wide association study (ML-GWAS) approaches identified 142 quantitative trait nucleotides (QTNs) in E1-E4 and the best linear unbiased prediction (BLUP), explaining 0.49-22.52% phenotypic variance. Of these, 38 novel stable QTNs were identified across at least two environments/methods, and their alleles showed significant TBW-DI differences. The number of superior alleles associated with TBW resistance for each accession ranged from 4 to 24; eight accessions had more than 18 superior alleles. Based on TBW-resistant alleles, the five best cross combinations were predicted, including MC133 × Ruyuan No. 1 and CO258 × ROX28. We identified 52 candidate genes around 38 QTNs related to TBW resistance based on homologous functional annotation and KEGG enrichment analysis, e.g., CYCD3;2, BSK1, Nitab4.5_0000641g0050, Nitab4.5_0000929g0030. To the best of our knowledge, this is the first comprehensive study to identify QTNs, superior alleles, and their candidate genes for breeding TBW-resistant tobacco varieties. The results provide further insight into the genetic architecture, marker-assisted selection, and functional genomics of TBW resistance, improving future breeding efforts to increase crop productivity.

[Nitrogen uptake and allocation characteristics of flue-cured tobacco in Nanxiong tobacco-planting area of Guangdong Province].

By the method of field in situ culture and 15N isotopic tracer technique, and taking flue-cured tobacco (Nicotiana tobacum) cultivar K326 as test material, a field experiment was conducted in the Nanxiong tobacco-planting area of Guangdong Province to study the characteristics of soil nitrogen (N) mineralization, the patterns of N accumulation and allocation in tobacco plants, and the allocation of plant-absorbed fertilizer N applied in current growth season. In the study area, the amount of soil mineralized N increased with tobacco growth, peaked at 75 days after transplanting, and decreased thereafter. The soil mineralized N at each tobacco growth stage was significantly higher in the control than in the N fertilization treatment. The N accumulation in tobacco plant organs was in the order of leaf > stalk > root. Tobacco plants mainly absorbed fertilizer N at rosette stage and topping stage, and mainly absorbed soil N at mature stage. The absorbed N in tobacco whole growth period was mainly derived from soil N, and the absorbed soil N and its proportion to the total absorbed N increased evidently with extending growth stage and ascending leaf position. The fertilizer N use efficiency per plant and the residual rate and loss rate of applied fertilizer N were 30. 8%, 32. 3% , and 36. 9% , respectively. In the study area, soil N mineralization rate was relatively high, and soil N had greater effects on the quality of upper tobacco leaves. Under the application rate of 150 kg N x hm(-2), the residual amount and loss amount of applied fertilizer N were relatively high.