Transcriptomics and iTRAQ-proteomics analyses provide novel insights into the defense mechanism of black shank disease in tobacco.

Black shank disease caused by Phytophthora nicotianae is one of the most important diseases in tobacco worldwide and can result in a devastating loss in tobacco cultivation. Many efforts have been carried out to identify the chromosome segment from Nicotiana plumbaginifolia containing a resistance locus carrying a gene named Php; however, the Php gene has not been cloned, and knowledge of the potential mechanism of the Php gene in the resistant lines is limited. To further characterize the resistance mechanism of the Php gene, we first used the resistant line "RBST" and the susceptible cultivar "Honghuadajinyuan" (HD) to obtain the near-isogenic line RBS89 containing the Php gene from RBST. RBS89 showed high resistance to black shank disease. Transcriptomic and iTRAQ analyses were applied to explore the potential defense mechanisms in RBS89 plants in comparison with HD plants with or without inoculation. Many differentially expressed genes (DEGs) and proteins were identified, and some pathogenesis-related (PR) proteins were extensively abundant in the RBS89 plants when compared with the HD plants in response to black shank disease. Importantly, overexpression of the PR gene NtPR-1B in HD plants improved the resistance of tobacco plants to black shank disease, indicating that NtPR-1B and Php genes might have similar roles in protecting tobacco from black shank disease. However, the relationship between NtPR-1B and Php genes requires further analysis. Therefore, our study provides valuable information for breeding tobacco cultivars with black shank disease resistance and sheds light on the defense mechanism of black shank disease in tobacco for enhancing Phytophthora resistance in other Solanaceae crops.

Transcriptomic profile of tobacco in response to Phytophthora nicotianae infection.

Black shank, caused by Phytophthora nicotianae (P. nicotianae), is a serious disease of cultivated tobacco (Nicotiana tabacum) worldwide. The interactions between tobacco and P. nicotianae are complex and the outcomes of the interactions depend on the tobacco genotype, P. nicotianae strain, and environmental conditions. In this study, we used RNA-sequencing (RNA-Seq) to investigate and compare transcriptional changes in the stems of tobacco upon inoculation with P. nicotianae strain race 0. We used two tobacco varieties: RBST (named from resistance to black shank and tobacco mosaic virus), which was resistant to the P. nicotianae strain race 0, and Honghuadajinyuan (HD), which was susceptible to P. nicotianae race 0. Samples were collected 12 and 72-hour post inoculation (hpi). Analysis of differentially expressed genes (DEGs) and significantly enriched GO terms indicated that several basic defense mechanisms were suppressed in both varieties, which included response to wounding (GO: 0009611), and defense response to fungus (GO: 0050832). We also found some genes that may especially be related to mechanisms of resistance in RBST, such as the one encoding a chitinase. These results will provide a valuable resource for understanding the interactions between P. nicotianae and tobacco plants.

iTRAQ-based quantitative proteomic analysis reveals proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum) in response to drought stress.

Drought is one of the most severe forms of abiotic stresses that threaten the survival of plants, including crops. In turn, plants dramatically change their physiology to increase drought tolerance, including reconfiguration of proteomes. Here, we studied drought-induced proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum), a solanaceous plant, using the isobaric tags for relative and absolute quantitation (iTRAQ)-based protein labeling technology. Of identified 5570 proteins totally, drought treatment increased and decreased abundance of 260 and 206 proteins, respectively, compared with control condition. Most of these differentially regulated proteins are involved in photosynthesis, metabolism, and stress and defense. Although abscisic acid (ABA) levels greatly increased in drought-treated tobacco leaves, abundance of detected ABA biosynthetic enzymes showed no obvious changes. In contrast, heat shock proteins (HSPs), thioredoxins, ascorbate-, glutathione-, and hydrogen peroxide (H2O2)-related proteins were up- or down-regulated in drought-treated tobacco leaves, suggesting that chaperones and redox signaling are important for tobacco tolerance to drought, and it is likely that redox-induced posttranslational modifications play an important role in modulating protein activity. This study not only provides a comprehensive dataset on overall protein changes in drought-treated tobacco leaves, but also shed light on the mechanism by which solanaceous plants adapt to drought stress.

Diversity arrays technology (DArT) for studying the genetic polymorphism of flue-cured tobacco (Nicotiana tabacum).

Diversity arrays technology (DArT) is a microarray-based marker system that achieves high throughput by reducing the complexity of the genome. A DArT chip has recently been developed for tobacco. In this study, we genotyped 267 flue-cured cultivars/landraces, including 121 Chinese accessions over five decades from widespread geographic regions in China, 103 from the Americas, and 43 other foreign cultivars, using the newly developed chip. Three hundred and thirty polymorphic DArT makers were selected and used for a phylogenetic analysis, which suggested that the 267 accessions could be classified into two subgroups, which could each be further divided into 2‒4 sections. Eight elite cultivars, which account for 83% of the area of Chinese tobacco production, were all found in one subgroup. Two high-quality cultivars, HHDJY and Cuibi1, were grouped together in one section, while six other high-yield cultivars were grouped into another section. The 330 DArT marker clones were sequenced and close to 95% of them are within non-repetitive regions. Finally, the implications of this study for Chinese flue-cured tobacco breeding and production programs were discussed.

[Genetic and correlation analysis for agronomic traits in flue-cured tobacco (Nicotiana tabacum L.)].

By using a genetic model including additive and dominance effects and their interaction with environments, 7 agronomic traits were analyzed for a diallel design in 4 environmental conditions with 14 flue-cured tobacco varieties (or breeding lines) and their 41 F1 crosses. It was revealed that additive effects were the major genetic component for plant height, internode length, and width of leaves. Number of leaves and length of leaves were mainly controlled by dominance x environment interaction effects. Additive x environment interaction effects and dominance x environment interaction effects played a major role for girth of stem. Yield performance was mainly controlled by additive effects and dominance x environment interaction effects. The varieties adapted to local environments tended to have highly positive additive effects. Dominance effects and the dominance x environment interaction effects could perform differently in positive or negative direction for many crosses. The breeding program for hybrids should consider the adaptation of hybrids to specific ecological environments. The analysis of correlation among agronomic traits indicated that most of phenotypic, genotypic, additive and dominance correlation coefficients were positive. Additive correlations were predominance in genetic correlations for most pairs of traits. Yield can be improved by indirect selection on plant height.

[Genetic diversity of flue-cured tobacco varieties based on ISSR markers].

Genetic diversity was assessed among 24 flue-cured tobacco varieties by ISSR (inter simple sequence repeats). A total of 100 ISSR primers were used to amplify the DNA from these varieties, of which 10 primers produced reproducible amplified products. Using polyacrylamide gel electrophoresis 208 bands were identified, of which 141 bands were polymorphic among the flue-cured tobacco varieties analyzed. Each primer produced 7-37 bands, the length of which ranged 200-2,400 bp. The ratio of polymorphic bands (PPB) was 67.79%. By cluster analysis based on ISSR markers using UPGMA, 24 varieties were divided into 5 major groups, in which the biggest group consisted of 12 varieties derived from Coker319. The genetic similarity index was 0.66-0.85 among 24 flue-cured tobacco varieties. Low genetic diversity among flue-cured tobacco varieties suggested that it is necessary to expand the genetic base of the flue-cured tobacco. 24 varieties could be distinguished by using 2 ISSR markers. The result also indicated that ISSR analysis was suitable for varietal identification and the study on genetic diversity of tobacco germplasm.

[Genetic contribution of agronomic traits to yield in flue-cured tobacco (Nicotiana tabacum L.)].

In order to understand the genetic contribution of six agronomic traits to yield, 14 flue-cured tobacco varieties (or breeding lines) and their 41 F1 crosses were used for multivariable conditional analysis. The contribution of additive variance of plant height to yield was larger than other agronomic traits. The largest contribution of dominant variance to yield was due to the length of middle leaves. All agronomic traits investigated had small contribution to yield due to additive x environment interaction effects and dominant x environment interaction effects. No identical trait of different parents showed the largest contribution to additive effect of yield. This could be resulted from the fact that each parent had its own genetic and developmental characterization. The dominant effects of yield were mainly influenced by length of middle leaves in most crosses. Length of middle leaves could be served as ameasurement to indirectly select the cross parent having high dominant effect of yield.