screening-of-tobacco-genotypes-for-phytophthora-nicotianae-resistance

Screening of Tobacco Genotypes for <em>Phytophthora nicotianae</em> Resistance

Black shank, caused by the oomycetes Phytophthora nicotianae, is destructive to tobacco, and this pathogen is highly pathogenic to many solanaceous crops. ...

Yunnan Academy of Tobacco Agricultural Sciences

The interaction between noncoding endogenous target mimicry (eTM) and its corresponding microRNA (miRNA) is a newly discovered regulatory mechanism and plays pivotal roles in various biological processes in plants. Tobacco (Nicotiana tabacum) is a model plant for studying secondary metabolite alkaloids, of which nicotine accounts for approximately 90%. In this work, we identified four unique tobacco-specific miRNAs that were predicted to target key genes of the nicotine biosynthesis and catabolism pathways and an eTM, novel tobacco miRNA (nta)-eTMX27, for nta-miRX27 that targets QUINOLINATE PHOSPHORIBOSYLTRANSFERASE2 (QPT2) encoding a quinolinate phosphoribosyltransferase. The expression level of nta-miRX27 was significantly down-regulated, while that of QPT2 and nta-eTMX27 was significantly up-regulated after topping, and consequently, nicotine content increased in the topping-treated plants. The topping-induced down-regulation of nta-miRX27 and up-regulation of QPT2 were only observed in plants with a functional nta-eTMX27 but not in transgenic plants containing an RNA interference construct targeting nta-eTMX27. Our results demonstrated that enhanced nicotine biosynthesis in the topping-treated tobacco plants is achieved by nta-eTMX27-mediated inhibition of the expression and functions of nta-miRX27. To our knowledge, this is the first report about regulation of secondary metabolite biosynthesis by an miRNA-eTM regulatory module in plants. 

Regulation of Nicotine Biosynthesis by an Endogenous Target Mimicry of MicroRNA in Tobacco.

Roles of microRNAs (miRNAs) and short interfering RNAs (siRNAs) in biotic stress responses, e.g., viral infection, have been demonstrated in plants by many studies. Tomato yellow leaf curl China virus (TYLCCNV) is a monopartite begomovirus that can systemically infect Solanaceae plants, and induces leaf curling, yellowing and enation symptoms when co-inoculated with a betasatellite (TYLCCNB). The released genome sequence of Nicotiana benthamiana provides an opportunity to identify miRNAs and siRNAs responsive to begomovirus-associated betasatellite in N. benthamiana.

A diverse set of miRNAs responsive to begomovirus-associated betasatellite in Nicotiana benthamiana.

Currently, developing genetic linkage map mostly use the derived-populations from crossing of two homogenous parents, which only covers limited genetic diversity and is inappropriate for some species, such as tobacco with lower diversity in genome. It is very general that there are no sufficient polymorphic markers to construct linkage map and ineffective to conduct marker-assisted selection (MAS) and quantitative trait locus (QTL) mapping based on lower density linkage map. This study proposed a method for developing genetic linkage map based on a four-way cross population. Computer simulation was conducted to investigate the feasibility and effectiveness of the method and a supporting program was designed. The main procedures and features of the proposed method were summarized as follows: 1) estimating genetic distance of any paired markers based on maximum likelihood method; 2) splitting all markers into different groups (linkage group) by cluster analysis based on genetic distance of markers; 3) for each linkage group, two end markers were first determined, then the marker order could be determined by inserting other markers in appropriate position by distance analysis of any three neighboring markers. Monte Carlo simulation showed that the proposed method is feasible, effective, and applicable in other derived populations from crossing of two homogenous parents.

Constructing linkage map based on a four-way cross population.

Black shank is a severe plant disease caused by the soil-borne pathogen Phytophthora nicotianae. Two physiological races of P. nicotianae, races 0 and 1, are predominantly observed in cultivated tobacco fields around the world. Race 0 has been reported to be more aggressive, having a shorter incubation period, and causing worse root rot symptoms, while race 1 causes more severe necrosis. The molecular mechanisms underlying the difference in virulence between race 0 and 1 remain elusive.

Genomes and virulence difference between two physiological races of Phytophthora nicotianae.

Tobacco (Nicotiana tabacum L.), particularly flue-cured tobacco, is one of the most economically important nonfood crops and is also an important model system in plant biotechnology. Despite its importance, only limited molecular marker resources are available for genome analysis, genetic mapping, and breeding. Simple sequence repeats (SSR) are one of the most widely-used molecular markers, having significant advantages including that they are generally co-dominant, easy to use, abundant in eukaryotic organisms, and produce highly reproducible results. In this study, based on the genome sequence data of flue-cured tobacco (K326), we developed a total of 13,645 mostly novel SSR markers, which were working in a set of eighteen tobacco varieties of four different types. A mapping population of 213 backcross (BC1) individuals, which were derived from an intra-type cross between two flue-cured tobacco varieties, Y3 and K326, was selected for mapping. Based on the newly developed SSR markers as well as published SSR markers, we constructed a genetic map consisting of 626 SSR loci distributed across 24 linkage groups and covering a total length of 1120.45 cM with an average distance of 1.79 cM between adjacent markers, which is the highest density map of flue-cured tobacco till date. 

Large-scale development of SSR markers in tobacco and construction of a linkage map in flue-cured tobacco.

Tomato zonate spot virus (TZSV), a dominant species of thrips-transmitted orthotospoviruses in Yunnan and Guangxi provinces in China, causes significant loss of yield in lots of crops and is a major threat to incomes of rural families. However, the detailed molecular mechanism of crop disease caused by TZSV remains obscure.

Transcriptomic profile of tobacco in response to Tomato zonate spot orthotospovirus infection.

Strigolactones (SLs) are a class of phytohormones that regulate plant architecture.  () genes are involved in the biosynthesis of SLs and are identified and characterized in many plants. However, the function of  genes in tobacco remains poorly understood. In this study, two closely related genes  and  were cloned from tobacco ( L.). The two  genes are orthologues of the tomato ()  8 () gene.  and  were primarily expressed in tobacco roots, but low expression levels of these genes were detected in all plant tissues, and their transcript levels significantly increased in response to phosphate limitation.  and  mutations were introduced into tobacco using the CRISPR/Cas9 system and transgenic tobacco lines for both  mutant alleles were identified. The  and  mutant alleles were inactivated by a deletion of three nucleotides and insertion of one nucleotide, respectively, both of which led to the production of premature stop codons. The  mutants had increased shoot branching, reduced plant height, increased number of leaves and nodes, and reduced total plant biomass compared to wild-type plants; however, the root-to-shoot ratio was unchanged. In addition, mutant lines had shorter primary roots and more of lateral roots than wild type. These results suggest that  genes are important for changes in tobacco plant architecture.

CRISPR/Cas9-Mediated Mutagenesis of Carotenoid Cleavage Dioxygenase 8 (CCD8) in Tobacco Affects Shoot and Root Architecture.

Advances in genomics technologies are making it increasingly feasible to characterize breeding lines that carry traits of agronomic interest. Tobacco germplasm lines that carry loci designated VAM and va have been extensively investigated due to their association with potyvirus resistance (both VAM and va) and defects in leaf surface compounds originating from glandular trichomes (VAM only). Molecular studies and classical genetic analyses are consistent with the model that VAM and va represent deletion mutations in the same chromosomal region. In this study, we used RNA-seq analysis, together with emerging tobacco reference genome sequence information to characterize the genomic regions deleted in tobacco lines containing VAM and va.

Characterization of Nicotiana tabacum genotypes possessing deletion mutations that affect potyvirus resistance and the production of trichome exudates.

The study performed genome-wide identification, characterization and evolution analysis of gene clusters for phytoalexin terpenoid biosynthesis in tobacco, and specifically illustrated ones for capsidiol, an efficient defensive specialized metabolite. Terpenoid phytoalexins play an important role in plant self-defense against pest and pathogen attack. Terpenoid biosynthesis involves terpene synthase and cytochrome P450, which always locate and function as cluster(s). In this study, we performed genome-wide investigation of metabolic gene clusters involved in terpenoid production in tobacco (Nicotiana tabacum). Due to the complexity of the tobacco genome, we modified a published prediction pipeline to reduce the influence of the large number of repeats and to improve the annotation of tobacco genes with respect to their metabolic functions. We identified 1181 metabolic gene clusters with 34 of them potentially being involved in terpenoid biosynthesis. Through integration with transcriptome and metabolic pathway annotation analyses, 3 of the 34 terpenoid biosynthesis-related gene clusters were determined to be high-confidence ones, with 2 involved in biosynthesis of capsidiol, a terpenoid recognized as 1 of the effective resistance compounds in the Nicotiana species. The capsidiol-related gene cluster was conserved in N. sylvestris, N. tomentosiformis and N. attenuate. Our findings demonstrate that phytoalexins in tobacco can arise from operon-like gene clusters, a genomic pattern characterized as being beneficial for rapid stress response, gene co-regulation, co-function and co-heredity.

Characterization and evolution of gene clusters for terpenoid phytoalexin biosynthesis in tobacco.

MADS-box genes play a pivotal role in various processes, including floral and seed development, controlling flowering time, regulation of fruits ripening, and respond to abiotic and biotic stressors in planta. Tobacco () has been widely used as a model plant for analyzing the gene function, however, there has been less information on the regulation of flowering, and the associated genes. In the present study, a total of 168 NtMADS-box genes were identified from tobacco, and their phylogenetic relationship, chromosome locations, and gene structures were further analyzed. NtMADS-box genes can be clustered into four sub-families of Mα, Mγ, MIKC*, and MIKC. A total of 111 NtMADS-box genes were distributed on 20 chromosomes, and 57 NtMADS-box genes were located on the unanchored scaffolds due to the complex and incomplete assembly of the tobacco genome. Expression profiles of NtMADS-box genes by microarray from 23 different tissues indicated that members in different NtMADS-box gene subfamilies might play specific roles in the growth and flower development, and the transcript levels of 24 NtMADS-box genes were confirmed by quantitative real-time PCR. Importantly, overexpressed / could promote early flowering and dwarfism in transgenic tobacco plants. Therefore, our findings provide insights on the characterization of NtMADS-box genes to further study their functions in plant development.

Genome-Wide Identification, Gene Structure and Expression Analysis of the MADS-Box Gene Family Indicate Their Function in the Development of Tobacco ().

Non-coding RNAs (ncRNAs), including microRNAs (miRNAs), long ncRNAs (lncRNAs) and circular RNAs (circRNAs), accomplish remarkable variety of biological functions. However, the composition of ncRNAs and their interactions with coding RNAs in modulating and controlling of cellular process in plants is largely unknown. Using a diverse group of high-throughput sequencing strategies, the mRNA, miRNA, lncRNA and circRNA compositions of tobacco (Nicotiana tabacum) roots determined and their alteration and potential biological functions in response to topping treatment analyzed.

A transcriptomic profile of topping responsive non-coding RNAs in tobacco roots (Nicotiana tabacum).

Tobacco (Nicotiana tabacum L.) is an essential commercial crop and an ideal model plant for biological mechanism studies. As an allopolyploid species, tobacco harbors a massive and complex genome, which makes the application of molecular markers complicated and challenging. In our study, we performed whole-genome sequencing of an intraspecific recombinant inbred line (RIL) population, a F1 generation and their parents. With the Nicotiana tabacum (K326 cultivar) genome as reference, a total of 45,081 markers were characterized to construct the genetic map, which spanned a genetic distance of 3486.78 cM. Evaluation of a two-dimensional heat map proved the high quality of the genetic map. We utilized these markers to anchor scaffolds and analyzed the ancestral genome origin of linkage groups (LGs). Furthermore, such a high-density genetic map will be applied for quantitative trait locus (QTL) detection, gene localization, genome-wide association studies (GWAS), and marker-assisted breeding in tobacco.

Construction of a high-density genetic map with whole genome sequencing in Nicotiana tabacum L.

Leaf chemistry traits are some of the key factors influencing tobacco quality, which can be significantly reduced by lower chemical components in cured leaf. To improve tobacco quality through breeding, genetic diversity analysis, population structure analysis, and genome-wide association studies were performed in a panel of 347 tobacco germplasms and the markers associated with five leaf chemistry traits, including total sugar (TS), reducing sugar (RS), total nitrogen (TN), nicotine (NIC), and total potassium (TP) contents were identified. Four groups were classified at a genetic distance of 0.316 by genetic diversity analysis based on coefficient parameter NEI72 using a program NTSYS-pc2.10e, whereas four well-differentiated subpopulations were postulated in the 347 tobacco accessions. A total of 47 target trait-associated SNPs was detected in at least three environments as well as the best linear unbiased predictions (BLUPs) across all environments, among which two, two, four, six, and one highly suggestive associated SNPs were repeatedly detected in all environments and BLUPs for TS, RS, TN, NIC, and TP, respectively. On the basis of the phenotypic effects of the alleles corresponding to suggestive associated SNPs, five tobacco accessions harboring favorable alleles with elite phenotypic performance in leaf chemistry traits were identified. The results could facilitate quality tobacco breeding for higher leaf chemistry trait contents through molecular marker-assisted approaches.

Genome-wide association study of leaf chemistry traits in tobacco.

The bHLH transcription factors play pivotal roles in plant growth and development, production of secondary metabolites and responses to various environmental stresses. Although the  genes have been well studied in model plant species, a comprehensive investigation of the  genes is required for tobacco with newly obtained high-quality genome. In the present study, a total of 309  genes were identified and can be divided into 23 subfamilies. The conserved amino acids which are essential for their function were predicted for the NtbHLH proteins. Moreover, the  genes were conserved during evolution through analyzing the gene structures and conserved motifs. A total of 265  genes were localized in the 24 tobacco chromosomes while the remained 44  genes were mapped to the scaffolds due to the complexity of tobacco genome. Moreover, transcripts of  genes were obviously tissue-specific expressed from the gene-chip data from 23 tobacco tissues, and expressions of 20 random selected  genes were further confirmed by quantitative real-time PCR, indicating their potential functions in the plant growth and development. Importantly, overexpressed  gene confers improve drought tolerance in tobacco indicating that it might be involved in the regulation of drought stress. Therefore, our findings here provide a valuable information on the characterization of  genes and further investigation of their functions in tobacco.

Genome-wide identification and expression analysis of NtbHLH gene family in tobacco () and the role of NtbHLH86 in drought adaptation.

 Vivianiis 1802 is an annual herb, native to Mexico and South America. It is one of the most widely distributed tobacco species. As a wild tobacco,  has provided several economically important disease-resistance genes to cultivated tobacco. We assembled the complete chloroplast genome of . The chloroplast genome is 155,945 bp in length, which includes a large single copy region (86,621 bp), a small single copy region (18,528 bp) and two separated inverted repeat regions (25,398 bp). A total of 117 unique genes were annotated, consisting of 84 protein-coding genes, 29 tRNA genes and 4 rRNA genes. Based on chloroplast genomes of 17  species, phylogenetic analyses indicated that  was closely related to  and .

Article	Year
Regulation of Nicotine Biosynthesis by an Endogenous Target Mimicry of MicroRNA in Tobacco. Plant physiology\| PubMed ID: 26246450	2015
A diverse set of miRNAs responsive to begomovirus-associated betasatellite in Nicotiana benthamiana. BMC plant biology\| PubMed ID: 24618068	2014
Constructing linkage map based on a four-way cross population. Zhejiang daxue xue bao. Nong ye yu sheng ming ke xue ban = Journal of Zhejiang University. Agriculture and life sciences\| PubMed ID: 25541573	2014
Genomes and virulence difference between two physiological races of Phytophthora nicotianae. GigaScience\| PubMed ID: 26823972	2016
Large-scale development of SSR markers in tobacco and construction of a linkage map in flue-cured tobacco. Breeding science\| PubMed ID: 27436948	2016
Transcriptomic profile of tobacco in response to Tomato zonate spot orthotospovirus infection. Virology journal\| PubMed ID: 28807054	2017
CRISPR/Cas9-Mediated Mutagenesis of Carotenoid Cleavage Dioxygenase 8 (CCD8) in Tobacco Affects Shoot and Root Architecture. International journal of molecular sciences\| PubMed ID: 29614837	2018
Characterization of Nicotiana tabacum genotypes possessing deletion mutations that affect potyvirus resistance and the production of trichome exudates. BMC genomics\| PubMed ID: 29925313	2018
Characterization and evolution of gene clusters for terpenoid phytoalexin biosynthesis in tobacco. Planta\| PubMed ID: 31414203	2019
Genome-Wide Identification, Gene Structure and Expression Analysis of the MADS-Box Gene Family Indicate Their Function in the Development of Tobacco (). International journal of molecular sciences\| PubMed ID: 31614589	2019
A transcriptomic profile of topping responsive non-coding RNAs in tobacco roots (Nicotiana tabacum). BMC genomics\| PubMed ID: 31726968	2019
Construction of a high-density genetic map with whole genome sequencing in Nicotiana tabacum L. Genomics\| PubMed ID: 31760041	2020
Genome-wide association study of leaf chemistry traits in tobacco. Breeding science\| PubMed ID: 32714047	2020
Genome-wide identification and expression analysis of NtbHLH gene family in tobacco () and the role of NtbHLH86 in drought adaptation. Plant diversity\| PubMed ID: 35024520	2021
The complete chloroplast genome of . Mitochondrial DNA. Part B, Resources\| PubMed ID: 35087940	2022

Bingguang Xiao

Biography

JoVE Journal Publications

Other Publications