Metabolic Profiling of Resistant and Susceptible Tobaccos Response Incited by Ralstonia pseudosolanacearum Causing Bacterial Wilt

The causal agent of bacterial wilt, Ralstonia pseudosolanacearum, can cause significant economic losses during tobacco production. Metabolic analyses are a useful tool for the comprehensive identification of plant defense response metabolites. In this study, a gas chromatography-mass spectrometry (GC-MS) approach was used to identify metabolites differences in tobacco xylem sap in response to R. pseudosolanacearum CQPS-1 in two tobacco cultivars: Yunyan87 (susceptible to R. pseudosolanacearum) and K326 (quantitatively resistant). Metabolite profiling 7 days post inoculation with R. pseudosolanacearum identified 88 known compounds, 42 of them enriched and 6 depleted in the susceptible cultivar Yunyan87, while almost no changes occurred in quantitatively resistant cultivar K326. Putrescine was the most enriched compound (12-fold) in infected susceptible tobacco xylem, followed by methyl-alpha-d-glucopyranoside (9-fold) and arabinitol (6-fold). Other sugars, amino acids, and organic acids were also enriched upon infection. Collectively, these metabolites can promote R. pseudosolanacearum growth, as shown by the increased growth of bacterial cultures supplemented with xylem sap from infected tobacco plants. Comparison with previous metabolic data showed that beta-alanine, phenylalanine, and leucine were enriched during bacterial wilt in both tobacco and tomato xylem.

Identification of two novel poleroviruses and the occurrence of Tobacco bushy top disease causal agents in natural plants

Tobacco bushy top disease (TBTD) is a devastating tobacco disease in the southwestern region of China. TBTD in the Yunnan Province is often caused by co-infections of several plant viruses: tobacco bushy top virus (TBTV), tobacco vein distorting virus (TVDV), tobacco bushy top virus satellite RNA (TBTVsatRNA) and tobacco vein distorting virus-associated RNA (TVDVaRNA). Through this study, two new poleroviruses were identified in two TBTD symptomatic tobacco plants and these two novel viruses are tentatively named as tobacco polerovirus 1 (TPV1) and tobacco polerovirus 2 (TPV2), respectively. Analyses of 244 tobacco samples collected from tobacco fields in the Yunnan Province through RT-PCR showed that a total of 80 samples were infected with TPV1 and/or TPV2, and the infection rates of TPV1 and TPV2 were 8.61% and 29.51%, respectively. Thirty-three TPV1 and/or TPV2-infected tobacco samples were selected for further test for TBTV, TVDV, TBTVsatRNA and TVDVaRNA infections. The results showed that many TPV1 and/or TPV2-infected plants were also infected with two or more other assayed viruses. In this study, we also surveyed TBTV, TVDV, TBTVsatRNA and TVDVaRNA infections in a total of 1713 leaf samples collected from field plants belonging to 29 plant species in 13 plant families and from 11 provinces/autonomous regions in China. TVDV had the highest infection rates of 37.5%, while TVDVaRNA, TBTV and TBTVsatRNA were found to be at 23.0%, 12.4% and 8.1%, respectively. In addition, TVDV, TBTV, TBTVsatRNA and TVDVaRNA were firstly detected of co-infection on 10 plants such as broad bean, pea, oilseed rape, pumpkin, tomato, crofton weed etc., and 1 to 4 of the TBTD causal agents were present in the samples collected from Guizhou, Hainan, Henan, Liaoning, Inner mongolia and Tibet autonomous regions. The results indicated that TBTD causal agents are expanding its host range and posing a risk to other crop in the field.

A Survey of Five Plant Viruses in Weeds and Tobacco in Poland

Weeds may contribute to the spread of plant virus epidemics by acting as reservoirs of viruses or/and their vectors. The aim of this research was to study the prevalence of five viral pathogens in weeds in the fields of solanaceous crops in six provinces in Poland differing with soil and climate conditions. Most of the sampled sites were associated with tobacco production. The total number of 157 samples of tobacco and 600 samples of weeds were subjected to DAS-ELISA detection of tomato spotted wilt orthotospovirus (TSWV), cucumber mosaic virus (CMV), potato virus Y (PVY), tobacco mosaic virus (TMV) and tobacco ringspot virus (TRSV). Twenty nine percent of samples of weeds were infected with at least one virus. TSWV and TMV were the most frequently detected in 17.5% and 14.7% of samples, respectively. In most provinces where infected tobacco was found, the same virus was also detected in weeds. Results of this survey are discussed in the context of the current status of virus epidemics in tobacco fields in Poland.

Intergrated metagenomics and metabolomics analysis discovers nematicidal microbes, enzymes and metabolites from the plant rhizosphere microbiota

Background: Root-knot nematode Meloidogyne incognita infects root systems of many crops resulting in huge decrease of crop production. Nematicidal microorganisms provides a safe and effective strategy to control M. incognita infection. In order to find microorganisms with high activity and new nematicidal metabolites, we collected the M. incognita infected tobacco rhizosphere soils (RNI) and non-infected tobacco rhizosphere soils (NS), and investigated their microbial community and network via metagenomics and metabolomics analysis. Results: Microbial networks of RNI soils were very different from the NS soils. Many nematicidal microorganisms were enriched in the NS soils, including isolates of Aspergillus , Achromobacter , Acinetobacter , Bacillus , Burkholderia , Comamonas , Enterobacter , Lysobacter , Microbacterium , Paenibacillus , Pantoea , Pseudomonas , Streptomyces and Variovorax. Enzymes analysis showed these nematicidal microorganisms can produce proteases, chitinase and lipases. The functions genes belonging to pathways of secondary metabolites biosynthesis and carbohydrate transport and metabolism were overrepresented in the rhizophere microbiota of NS soils comparing with the RNI soils. 102 metabolites contents were significantly different between the RNI and NS rhizosphere microbiota. 35 metabolites were overrepresented in the NS soils comparing the RNI samples, including acetophenone. Acetophenone showed high nematicidal (LC 50 = 0.66 μg/ml) and avoidance activity against M. incognita . Bacillus amyloliquefaciens W1 could produce acetophenone. Liquid culture of W1 could kill 98.8% of M . incognita J2 juveniles after treatment for 24 h.Conclusions: In general, the rhizophere microbiota of NS soils could produce volatile materials, multiple enzymes and secondary metabolites against nematode. Collectively, the microbiota of NS and RNI rhizophere differed significantly in microbial network structure, community composition, function genes and metabolites. Collectively, combination of multi-omics analysis and culture-dependent technology is powerful for finding nematicidal microorganisms and metabolites from soil.

Research on the Interaction Mechanism Between α Mino-Phosphonate Derivative Q-R and Harpin-Binding Protein 1 in Tobacco (Nicotiana tabacum) Plants

Amino-phosphonate derivative R-diphenyl-1-(4-methylbenzothiazole-2-amino)-1-(thiphene-2-yl)-methylphosphonate (Q-R) has a high protective anti-tobacco mosaic virus (TMV) activity. However, the mechanism responsible for Q-R’s effect on TMV infection is largely unknown. Here, we studied the expression levels of harpin-binding protein 1 (HrBP1) and pathogenesis-related protein-1a (PR-1a) in TMV-infected tobacco plants by using reverse transcription quantitative real-time PCR. Then, we verified the interactions between Q-R and the HrBP1 protein from Escherichia coli using isothermal titration calorimetry and studied the Q-R-associated assembly of HrBP1 using size-exclusion chromatography. The results showed that the expression levels of HrBP1 and PR-1a genes were significantly increased by Q-R at the transcriptional level in TMV-infected tobacco plants, and the E. coli-expressed HrBP1 protein was assembled into oligomers by Q-R via binding to HrBP1 with a dissociation constant of 1.19 μM. We, therefore, concluded that Q-R activated the HrBP1 and PR-1a genes and enhanced the ability of HrBP1 to assemble in tobacco plants.

Different potato virus Y strains frequently co-localize in single epidermal leaf cells and in the aphid stylet

Single aphids can simultaneously or sequentially acquire and transmit multiple potato virus Y (PVY) strains. Multiple PVY strains are often found in the same field and occasionally within the same plant, but little is known about how PVY strains interact in plants or in aphid stylets. Immuno-staining and confocal microscopy were used to examine the spatial and temporal dynamics of PVY strain mixtures (PVYO and PVYNTN or PVYO and PVYN) in epidermal leaf cells of ‘Samsun NN’ tobacco and ‘Goldrush’ potato. Virus binding and localization was also examined in aphid stylets following acquisition. Both strains systemically infected tobacco and co-localized in cells of all leaves examined; however, the relative amounts of each virus changed over time. Early in the tobacco infection, when mosaic symptoms were observed, PVYO dominated the infection although PVYNTN was detected in some cells. As the infection progressed and vein necrosis developed, PVYNTN was prevalent. Co-localization of PVYO and PVYN was also observed in epidermal cells of potato leaves with most cells infected with both viruses. Furthermore, two strains could be detected binding to the distal end of aphid stylets following virus acquisition from a plant infected with a strain mixture. These data are in contrast with the traditional belief of spatial separation of two closely related potyviruses and suggest apparent non-antagonistic interaction between PVY strains that could help explain the multitude of emerging recombinant PVY strains discovered in potato in recent years.

Intergrated Metagenomics and Metabolomics Analysis Discovers Nematicidal Microbes, Enzymes and Metabolites From the Plant Rhizosphere Microbiota

BackgroundRoot-knot nematode Meloidogyne incognita infects root systems of many crops resulting in huge decrease of crop production. Nematicidal microorganisms provides a safe and effective strategy to control M. incognita infection. In order to find more microorganisms with high activity and new nematicidal metabolites, we collected the M. incognita infected tobacco rhizosphere soils (RNI) and non-infected tobacco rhizosphere soils (NS), and investigated their microbial community and network via metagenomics and metabolomics analysis. ResultsMicrobial networks of RNI soils were very different from the NS soils. Many nematicidal microorganisms were enriched in the NS soils, including some isolates such as Aspergillus , Achromobacter , Acinetobacter , Bacillus , Burkholderia , Comamonas , Enterobacter , Lysobacter , Microbacterium , Paenibacillus , Pantoea , Pseudomonas , Streptomyces and Variovorax. Enzymes analysis showed these nematicidal microorganisms can produce proteases, chitinase and lipases. The functions genes belonging to pathways of secondary metabolites biosynthesis and carbohydrate transport and metabolism were overrepresented in the rhizophere microbiota of NS soils comparing with the RNI soils. 102 metabolites contents were significantly different between the RNI and NS rhizosphere microbiota. 35 metabolites were overrepresented in the NS soils comparing the RNI samples, including acetophenone. Acetophenone showed high nematicidal (LC 50 = 0.66 μg/ml) and avoidance activity against M. incognita . A isolate of Bacillus amyloliquefaciens W1 with production of acetophenone can kill 98.8% of M . incognita . ConclusionsIn general, the rhizophere microbiota of NS soils could produce volatile materials, multiple enzymes and secondary metabolites against nematode. Collectively, the microbiota of NS and RNI rhizophere differed significantly in microbial network structure, community composition, function genes and metabolites. Collectively, combination of multi-omics analysis and culture-dependent technology is powerful for finding nematicidal microorganisms and metabolites from soil.

Proteomic and Phosphoproteomic Analysis in Tobacco Mosaic Virus-Infected Tobacco (Nicotiana tabacum)

Tobacco mosaic virus (TMV) is a common source of biological stress that significantly affects plant growth and development. It is also useful as a model in studies designed to clarify the mechanisms involved in plant viral disease. Plant responses to abiotic stress were recently reported to be regulated by complex mechanisms at the post-translational modification (PTM) level. Protein phosphorylation is one of the most widespread and major PTMs in organisms. Using immobilized metal ion affinity chromatography (IMAC) enrichment, high-pH C18 chromatography fraction, and high-accuracy mass spectrometry (MS), a set of proteins and phosphopeptides in both TMV-infected tobacco and control tobacco were identified. A total of 4905 proteins and 3998 phosphopeptides with 3063 phosphorylation sites were identified. These 3998 phosphopeptides were assigned to 1311 phosphoproteins, as some proteins carried multiple phosphorylation sites. Among them, 530 proteins and 337 phosphopeptides corresponding to 277 phosphoproteins differed between the two groups. There were 43 upregulated phosphoproteins, including phosphoglycerate kinase, pyruvate phosphate dikinase, protein phosphatase 2C, and serine/threonine protein kinase. To the best of our knowledge, this is the first phosphoproteomic analysis of leaves from a tobacco cultivar, K326. The results of this study advance our understanding of tobacco development and TMV action at the protein phosphorylation level.