Comparative transcriptome analysis of fiber tissues between Gossypium barbadense and Gossypium hirsutum could reveal the molecular mechanisms underlying high-quality fiber formation and identify candidate genes for fiber quality improvement. In this study, 759 genes were found to be strongly upregulated at the elongation stage in G. barbadense, which showed four distinct expression patterns (I–IV). Among them, the 346 genes of group IV stood out in terms of the potential to promote fiber elongation, in which we finally identified 42 elongation-related candidate genes by comparative transcriptome analysis between G. barbadense and G. hirsutum. Subsequently, we overexpressed GbAAR3 and GbTWS1, two of the 42 candidate genes, in Arabidopsis plants and validated their roles in promoting cell elongation. At the secondary cell wall (SCW) biosynthesis stage, 2275 genes were upregulated and exhibited five different expression profiles (I–V) in G. barbadense. We highlighted the critical roles of the 647 genes of group IV in SCW biosynthesis and further picked out 48 SCW biosynthesis-related candidate genes by comparative transcriptome analysis. SNP molecular markers were then successfully developed to distinguish the SCW biosynthesis-related candidate genes from their G. hirsutum orthologs, and the genotyping and phenotyping of a BC₃F₅ population proved their potential in improving fiber strength and micronaire. Our results contribute to the better understanding of the fiber quality differences between G. barbadense and G. hirsutum and provide novel alternative genes for fiber quality improvement.

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.

Callose plays an important role in plant development and in response to a wide range of biotic and abiotic stresses. However, the systematic identification of callose synthase (CalS), the major enzyme for callose biosynthesis, has been delayed in crops, especially in Solanaceae. In the current research, 18 CalS genes (NtCalS1–NtCalS18) were identified in Nicotiana tabacum and classified into four subfamilies. A comprehensive analysis of their physicochemical properties, gene structure, and evolutionary history highlighted their evolutionary conservation. We also identified a number of NtCalSs that responded to the infection with Phytophthora nicotianae and Ralstonia solanacearum, as well as to drought and cold treatments, by analyzing RNA-seq data. NtCalS1 and NtCalS12, a highly homologous gene pair, were selected to create mutants using the CRISPR-Cas9 technology for their drastic response to Phytophthora nicotianae infection as well as the strong expression levels in roots. The mutants with the simultaneous knockout of NtCalS1 and NtCalS12, compared with the control plants, displayed more resistance to tobacco black shank caused by Phytophthora nicotianae. Furthermore, the real-time quantitative PCR (qRT-PCR) assay showed that the knockout of NtCalS1 and NtCalS12 activated the signaling pathways mediated by plant hormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) before and after the infection of Phytophthora nicotianae and thus may have contributed to tobacco immunity against black shank. These findings contribute valuable information for further understanding the roles of CalS genes in tobacco stress responses and provide alternative genes for resistance improvement.