Development of user-friendly markers for disease resistance to black root rot of tobacco through genotyping by sequencing

Black root rot (BRR), a disease caused by the hemibiotrophic fungus Thielaviopsis basicola, seriously compromises yield and leaf quality in tobacco (Nicotiana tabacum). Full resistance to black root rot, conferred by the resistance to BRR 1 (RBRR1) locus from Nicotiana debneyi Domin, was transferred to a burley tobacco cultivar through interspecific hybridization. Some undesirable traits potentially caused by linkage drag restrict wider application of RBRR1 in flue-cured tobacco. Therefore, user-friendly molecular markers are needed to assist selection for resistance to black root rot and to break the unfavorable linkage. Genotyping by sequencing (GBS) is a rapid and robust approach for reduced representation sequencing of multiplexed genomic DNA samples that combines genome-wide molecular marker discovery with genotyping. In the present study, we used GBS to identify single-nucleotide polymorphisms (SNPs) linked to the RBRR1 locus, and PCR-based assays for detection of these SNPs were also developed. Sequence analysis of the SNP markers suggested that RBRR1 is located on chromosome 17, providing a basis for map-based cloning of this valuable gene. Co-dominant CAPS markers that co-segregate with the disease-resistant phenotype offer user-friendly tools for tobacco breeding and variety improvement. Furthermore, tested with diverse N. tabacum germplasm, SS192650 displayed 100% selection accuracy for resistance to BRR, suggesting that this marker can be used in diverse tobacco populations.     

<Emphasis Type="Italic">NmEXT</Emphasis> Extensin Gene: a Positive Regulator of Resistance Response Against the Oomycete <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis>

The oomycete pathogens produce important diseases in many plant species. To identify extensin genes expressed during the oomycete Phytophthora nicotianae-Nicotiana megalosiphon interaction, we used the SuperSAGE technology. Using this approach, we detected a N. megalosiphon extensin gene (NmEXT) triggered during the interaction. The extensin gene accumulation induced by the pathogen correlated with disease resistance in different Nicotiana species. Transient expression of NmEXT gene in susceptible Nicotiana tabacum enhanced the resistance to P. nicotianae. Our date indicated that NmEXT gene served a positive role in N. tabacum resistance against P. nicotianae.     

Integrated control of tobacco black shank by combined use of riboflavin and <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain Tpb55

We investigated the effect of riboflavin on the biocontrol activity of Bacillus subtilis Tpb55 against Phytophthora nicotianae (Pn), which causes tobacco black shank. Riboflavin (0.2 mg ml^?1) significantly improved the biocontrol activity of Tpb55 (2.0 × 10⁸ cfu ml^?1). Riboflavin (0.02–0.5 mg ml^?1) alone could not significantly inhibit Pn growth. However, it enhanced the B. subtilis population, both in vitro and in tobacco roots and significantly increased the activity of defense enzymes, peroxidase, catalase, superoxide dismutase, and β-1,3-glucanase, in the roots of B. subtilis-treated tobacco seedlings. Our results indicate that riboflavin can stimulate the growth of B. subtilis Tpb55 and induce resistance to Pn in tobacco plants. These findings should boost the prospects for practical application of B. subtilis Tpb55 as a biocontrol agent against black shank of tobacco.     

Heterosis,transmission genetics,and selection for increased growth rate in a <Emphasis Type="Italic">N. tabacum</Emphasis> × synthetic tobacco cross

Cultivated tobacco (Nicotiana tabacum L.) is a classic amphidiploid, and hybrids between this cultivated species and closely related diploid Nicotiana relatives often exhibit heterotic effects for growth rate and yield. Crosses between N. tabacum and synthetic tobaccos, 4x(Nicotiana sylvestris × Nicotiana otophora) or 4x(N. sylvestris × Nicotiana tomentosiformis), may provide superior routes for genome-wide introgression from diploid relatives and allow increased potential to capitalize on heterotic effects in tobacco. Significant levels of mid-parent heterosis were observed for yield and growth rate in F₁ hybrids between synthetic tobaccos and a standard tobacco cultivar. Microsatellite marker genotyping of an F₂ population derived from a K326 × [4x(N. sylvestris × N. otophora)] cross was carried out to preliminarily investigate the relative importance of different types of gene action on observed heterosis in the original interspecific cross. Results suggested a role for both partial dominance and overdominance. Marker genotyping also indicated an overall reduced level of recombination in the N. tabacum × synthetic tobacco cross relative to a N. tabacum × N. tabacum cross but no evidence of genomic regions with severely restricted levels of recombination. Results suggest that populations derived from N. tabacum × synthetic tobacco crosses may be more efficient for introgressing germplasm from diploid relatives, as compared to populations derived from crosses between N. tabacum and diploid forms where preferential pairing between N. tabacum homologues can reduce the potential for introgression of alien chromatin. Such materials may be useful as sources of favorable alleles influencing quantitative characters in tobacco.     

Transmission of RNA silencing signal through grafting confers virus resistance from transgenically silenced tobacco rootstocks to non-transgenic tomato and tobacco scions

We examined the transmission of RNA silencing signal in non-transgenic tomato and tobacco scions grafted onto the tobacco Sd1 rootstocks, which is silenced in both NtTOM1 and NtTOM3 required for tobamovirus multiplication. When the non-transgenic tomato scions were grafted onto the Sd1 rootstocks, RT-PCR analysis of the scions showed the reduced level of mRNA compared with that before grafting in both LeTH3 and LeTH1, tomato homologs of NtTOM1 and NtTOM3, respectively. siRNAs from both genes were detected in the scions after grafting but not before grafting. Further tomato scions were inoculated with Tomato mosaic virus (ToMV) and used for virus infection. They showed very low level of virus accumulation. Necrotic responding tobacco to tobamovirus was grafted onto the rootstock of Sdl. RT-PCR analysis showed low level expression of both NtTOM1 and NtTOM3 in the scions but siRNA was detected after grafting. When the leaves of scions were inoculated with ToMV or Tobacco mosaic virus, they produced very few local necrotic lesions (LNLs) while the control scions did many LNLs. These results suggest that RNA silencing was transmitted to non-transgenic tomato and tobacco scions after grafting onto the Sd1 rootstocks and that virus resistance was induced in the scions.     

The maize CorA/MRS2/MGT-type Mg transporter,ZmMGT10, responses to magnesium deficiency and confers low magnesium tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Key message

A new selectable marker gene for stable transformation of the plastid genome was developed that is similarly efficient as the aadA, and produces no background of spontaneous resistance mutants.

Abstract

More than 25 years after its development for Chlamydomonas and tobacco, the transformation of the chloroplast genome still represents a challenging technology that is available only in a handful of species. The vast majority of chloroplast transformation experiments conducted thus far have relied on a single selectable marker gene, the spectinomycin resistance gene aadA. Although a few alternative markers have been reported, the aadA has remained unrivalled in efficiency and is, therefore, nearly exclusively used. The development of new marker genes for plastid transformation is of crucial importance to all efforts towards extending the species range of the technology as well as to those applications in basic research, biotechnology and synthetic biology that involve the multistep engineering of plastid genomes. Here, we have tested a bifunctional resistance gene for its suitability as a selectable marker for chloroplast transformation. The bacterial enzyme aminoglycoside acetyltransferase(6′)-Ie/aminoglycoside phosphotransferase(2″)-Ia possesses an N-terminal acetyltransferase domain and a C-terminal phosphotransferase domain that can act synergistically and detoxify aminoglycoside antibiotics highly efficiently. We report that, in combination with selection for resistance to the aminoglycoside tobramycin, the aac(6′)-Ie/aph(2″)-Ia gene represents an efficient marker for plastid transformation in that it produces similar numbers of transplastomic lines as the spectinomycin resistance gene aadA. Importantly, no spontaneous antibiotic resistance mutants appear under tobramycin selection.

     

Characterization of harpin<Subscript>Xoo</Subscript> induced hypersensitive responses in non host plant,tobacco

Harpin proteins encoded by hrp genes are bacterial protein elicitors that can stimulate hypersensitive response (HR) in non-host plants. HR-related pathogen resistance involves a complex form of programmed cell death (PCD). It is increasingly viewed as a key component of the hypersensitive disease response of plants. Currently, the evidence of harpin proteins-induced PCD is deficient though it exhibits phenotypic parallels with HR, and the mechanism of harpin proteins-induced PCD is not well understood. In this study, we demonstrate that harpin_Xoo protein from Xanthomonas oryzae pv. oryzae of rice bacterial blight expressed and isolated from bacterial cells acted as an agent to induce PCD in infiltrated tobacco plants. Treatment of tobacco leaves with harpin_Xoo induced typical PCD-related morphological and biochemical changes including cell shrinkage and nuclear DNA degradation. We further analyzed the expression of several genes in signal transduction pathway of PCD in tobacco plants by real-time qRT-PCR analysis using EF-1α as an endogenous control. Our results showed that the expression of NtDAD1 was down-regulated and the expression of BI-1, tpa1 and aox1 was up-regulated following the infiltration of harpin_Xoo into tobacco leaves. Our data suggest that harpin_Xoo can induce PCD with the coordination of PCD-related genes in infiltrated tobacco leaves, providing evidence to further investigate the signal transduction pathways of HR and PCD.     

Heat resistance of the skeletal muscle in Western Palearctic green frogs (<Emphasis Type="Italic">Rana esculenta</Emphasis> complex)

Heat resistance of the gastrocnemius muscle was studied in five species of the Rana esculenta complex. It was similar in R. bedriagae, R. lessonae, and in the European form of R. ridibunda; while North African R. saharica demonstrated a lower heat resistance. No heterosis was expressed in R. esculenta, a clonal hybrid of R. lessonae and R. ridibunda, for the heat resistance of the muscle. Moreover, this species demonstrated low heat resistance at the highest test temperature (42°C). Comparison of diploid and triploid R. esculenta syntopically occurring in the same water bodies demonstrated no differences between them, thus, suggesting that polyploidy has no effect on this parameter at least in this case.     

Comparative studies of leaf surface chemical biosynthesis in different tobacco cultivars

To understand the differences in the biosynthesis of leaf surface chemicals and their influence on aphid preference for different tobacco cultivars (Nicotiana tabacum), we analyzed the secretory characteristics of glandular trichomes of four commercial cultivars, K326 (flue-cured), Beinhart 1000-1 (cigar), Basma YNOTBS1 (oriental), and Dabai 1 (burley), and their parental species, Nicotiana sylvestris and Nicotiana tomentosiformis. Trichome-type observation showed that K326 and N. sylvestris have three kinds of glandular trichomes (non-glandular, long stalked glandular, and short stalked glandular trichomes), whereas Beinhart 1000-1, Basma YNOTBS1, Dabai 1, and N. tomentosiformis had two kinds of glandular trichomes (long and short stalked glandular trichomes). The gas chromatography–mass spectrometry profiles of leaf exudates indicated that N. tomentosiformis synthesized only labdanoids; N. sylvestris, K326 and Dabai 1 synthesized only cembranoids; and Beinhart 1000-1 and Basma YNOTBS1 synthesized cembranoids and labdanoids. Gene expression pattern analysis revealed that the labdanoid synthesis-related genes NtABS and NtCPS2 were expressed in N. tomentosiformis, Beinhart 1000-1, and Basma YNOTBS1, whereas the cembranoid synthesis-related genes NtCYC and NtCYP71D16 were expressed in N. sylvestris and all four commercial cultivars. Evolutionary analysis indicated that NtCYC and NtCYP71D16 might be phylogenetically originated from N. sylvestris, whereas NtABS and NtCPS2 expressed in Basma YNOTBS1 and Beinhart 1000-1 might be derived from N. tomentosiformis. In addition, aphid attraction (number of aphids) was significantly and positively correlated with the total glandular secretion (r² =?0.9425, P?≤?0.05), and it was significantly and positively correlated with amount of CBT-diol (r²?=?0.9224; P?≤?0.05). These results provide new insights into the biosynthesis of diterpenoids and biotic stress resistance in tobacco.     

Overexpression of <Emphasis Type="Italic">IrlVHA</Emphasis>-<Emphasis Type="Italic">c</Emphasis>, A Vacuolar-Type H<Superscript>+</Superscript>-ATPase c Subunit Gene from <Emphasis Type="Italic">Iris lactea</Emphasis>, Enhances Salt Tolerance in Tobacco

Vacuolar-type H⁺-ATPase (V-ATPase), a multi-subunit endomembrane proton pump, plays an important role in plant growth and response to environmental stresses. In the present study, transgenic tobacco that overexpressed the V-ATPase c subunit gene from Iris lactea (IrlVHA-c) was used to determine the function of IrlVHA-c. Quantitative PCR analysis showed that IrlVHA-c expression was induced by salt stress in I. lactea roots and leaves. Subcellular localization of green fluorescent protein (GFP) as marker combined with FM4-64 staining showed that the IrlVHA-c-GFP was localized to the endosomal compartment in tobacco cells. Compared with the wild-type, the IrlVHA-c transgenic tobacco plants exhibited greater seed germination rates, root length, fresh weight, and higher relative water content (RWC) of leaves under salt stress. Furthermore, the IrlVHA-c transgenic tobacco leaves have lower stomatal densities and larger stomatal apertures than wild-type. Under salt stress, superoxide dismutase (SOD) activity in the transgenic tobacco was significantly enhanced. Moreover, the level of malondialdehyde (MDA) in the transgenic tobacco was significantly lower than that in wild-type plants under salt stress. Taken together, these results suggested that the IrlVHA-c plays an important role in salt tolerance in transgenic tobacco by influencing stomatal movement and physiological changes.