This study presents a novel approach of controlling vascular wilt in tomato by RNAi expression directed to pathogenicity genes of Fusarium oxysporum f. sp. lycopersici. Vascular wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici leads to qualitative and quantitative loss of the crop. Limitation in the existing control measures necessitates the development of alternative strategies to increase resistance in the plants against pathogens. Recent findings paved way to RNAi, as a promising method for silencing of pathogenicity genes in fungus and provided effective resistance against fungal pathogens. Here, two important pathogenicity genes FOW2, a Zn(II)2Cys6 family putative transcription regulator, and chsV, a putative myosin motor and a chitin synthase domain, were used for host-induced gene silencing through hairpinRNA cassettes of these genes against Fusarium oxysporum f. sp. lycopersici. HairpinRNAs were assembled in appropriate binary vectors and transformed into tomato plant targeting FOW2 and chsV genes, for two highly pathogenic strains of Fusarium oxysporum viz. TOFOL-IHBT and TOFOL-IVRI. Transgenic tomatoes were analyzed for possible attainment of resistance in transgenic lines against fungal infection. Eight transgenic lines expressing hairpinRNA cassettes showed trivial disease symptoms after 6-8 weeks of infection. Hence, the host-induced posttranscriptional gene silencing of pathogenicity genes in transgenic tomato plants has enhanced their resistance to vascular wilt disease caused by Fusarium oxysporum.

In Ralstonia solanacearum, a devastating phytopathogen whose metabolism is poorly understood, we observed that the Entner-Doudoroff (ED) pathway and nonoxidative pentose phosphate pathway (non-OxPPP) bypass glycolysis and OxPPP under glucose oxidation. Evidence derived from 13 C stable isotope feeding and genome annotation-based comparative metabolic network analysis supported the observations. Comparative metabolic network analysis derived from the currently available 53 annotated R. solanacearum strains, including a recently reported strain (F1C1), representing the four phylotypes, confirmed the lack of key genes coding for phosphofructokinase (pfk-1) and phosphogluconate dehydrogenase (gnd) enzymes that are relevant for glycolysis and OxPPP, respectively. R. solanacearum F1C1 cells fed with [ 13 C]glucose (99% [1-13 C]glucose or 99% [1,2-13 C]glucose or 40% [ 13 C 6 ]glucose) followed by gas chromatography-mass spectrometry (GC-MS)-based labeling analysis of fragments from amino acids, glycerol, and ribose provided clear evidence that rather than glycolysis and the OxPPP, the ED pathway and non-OxPPP are the main routes sustaining metabolism in R. solanacearum. The 13 C incorporation in the mass ions of alanine (m/z 260 and m/z 232), valine (m/z 288 and m/z 260), glycine (m/z 218), serine (m/z 390 and m/z 362), histidine (m/z 440 and m/z 412), tyrosine (m/z 466 and m/z 438), phenylalanine (m/z 336 and m/z 308), glycerol (m/z 377), and ribose (m/z 160) mapped the pathways supporting the observations. The outcomes help better define the central carbon metabolic network of R. solanacearum that can be integrated with 13 C metabolic flux analysis as well as flux balance analysis studies for defining the metabolic phenotypes. IMPORTANCE Understanding the metabolic versatility of Ralstonia solanacearum is important, as it regulates the trade-off between virulence and metabolism (1, 2) in a wide range of plant hosts. Due to a lack of clear evidence until this work, several published research papers reported on the potential roles of glycolysis and the oxidative pentose phosphate pathway (OxPPP) in R. solanacearum (3,4). This work provided evidence from 13 C stable isotope feeding and genome annotation-based comparative metabolic network analysis that the Entner-Doudoroff pathway and non-OxPPP bypass glycolysis and OxPPP during the oxidation of glucose, a component of the host xylem pool that serves as a potential carbon source (5). The outcomes help better define the central carbon metabolic network of R. solanacearum that can be integrated with 13 C metabolic flux analysis as well as flux balance analysis studies for defining the metabolic phenotypes. The study highlights the need to critically examine phytopathogens whose metabolism is poorly understood. on July 10, 2020 by guest http://msystems.asm.org/ Downloaded from R alstonia solanacearum is one of the most destructive plant pathogens as it infects over 450 plant species (6-8), and its metabolism is poorly understood. It is a soilborne pathogen that enters plan...

The shoot and fruit borer, Leucinodes orbonalis (Lepidoptera: Crambidae) is the major cause of low productivity in eggplant and insecticides being the mainstay of management of L. orbonalis. However, field control failures are widespread due to the evolution of insecticide resistance. Taking advantage of the whole genome sequence information, the present study investigated the level of insecticide resistance and the expression pattern of individual carboxylesterase (CE) and glutathione S-transferases (GSTs) genes in various field collected populations of L. orbonalis. Dose-mortality bioassays revealed a very high level of resistance development against fenvalerate (48.2–160-fold), phosalone (94-534.6-fold), emamectin benzoate (7.2–55-fold), thiodicarb (9.64–22.7-fold), flubendiamide (187.4–303.0-fold), and chlorantraniliprole (1.6–8.6-fold) in field populations as compared to laboratory-reared susceptible iso-female colony (Lo-S). Over-production of detoxification enzymes viz., CE and GST were evident upon enzyme assays. Mining of the draft genome of L. orbonalis yielded large number of genes potentially belonging to the CE and GST gene families with known history of insecticide resistance in other insects. Subsequent RT-qPCR studies on relative contribution of individual genes revealed over-expression of numerous GSTs and few CEs in field populations, indicating their possible involvement of metabolic enzymes in insecticide resistance. The genomic information will facilitate the development of novel resistance management strategies against this pest.

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In Ralstonia solanacearum, a devastating phytopathogen whose metabolism 13 is poorly understood, we observed that Entner-Doudoroff (ED) pathway and Oxidative pentose phosphate pathway (OxPPP) bypasses glycolysis and OxPPP under 15 glucose oxidation. Evidences derived from 13 C stable isotopes feeding and genome 16 annotation based comparative metabolic network analysis supported the observations. 17 Comparative metabolic network analysis derived from the currently available 53 18 annotated R. solanacearum strains also including the recently reported strain (F1C1), 19 representing the four phylotypes confirmed the lack of key genes coding for 20 phosphofructokinase (pfk-1) and phosphogluconate dehydrogenase (gnd) enzymes that 21 are relevant for glycolysis and OxPPP respectively. R. solanacearum F1C1 cells fed 22 with 13 C Glucose (99%[1-13 C]-or 99%[1,2-13 C]-or 40%[ 13 C 6 ]-glucose) followed by GC- 23MS based labelling analysis of fragments from amino acids, glycerol and ribose 24 provided clear evidence that rather than Glycolysis and OxPPP, ED pathway and Non-25 OxPPP are the main routes sustaining metabolism in R. solanacearum. The 13 C 26 incorporation in the mass ions of alanine (m/z 260, m/z 232); valine (m/z 288, m/z 260), 27 glycine (m/z 218), serine (m/z 390, m/z 362), histidine (m/z 440, m/z 412), tyrosine (m/z 28 466, m/z 438), phenylalanine (m/z 336, m/z 308), glycerol (m/z 377) and ribose (m/z 29 160) mapped the pathways supporting the observations. The outcomes help better 30 defining the central carbon metabolic network of R. solanacearum that can be 31 integrated with 13 C metabolic flux analysis as well as flux balance analysis studies for 32 defining the metabolic phenotypes. 33 Importance: Understanding the metabolic versatility of Ralstonia solanacearum is 37 important as it regulates the tradeoff between virulence and metabolism (1, 2) in a wide 38 range of plant hosts. Due to a lack of clear evidence until this work, several published 39 research papers reported on potential roles of Glycolysis and Oxidative pentose 40 phosphate pathways (OxPPP) in R. solanacearum (3, 4). This work provided evidence 41 from 13 C stable isotopes feeding and genome annotation based comparative metabolic 42 network analysis that Entner-Doudoroff pathway and Non-OxPPP bypasses glycolysis 43 and OxPPP during the oxidation of Glucose, one of the host xylem pool that serves as a 44 potential carbon source (5). The outcomes help better defining the central carbon 45 metabolic network of R. solanacearum that can be integrated with 13 C metabolic flux 46 analysis as well as flux balance analysis studies for defining the metabolic phenotypes.47The study highlights the need to critically examine phytopathogens whose metabolism 48 is poorly understood. 50Introduction: Ralstonia solanacearum is one of the most destructive plant pathogen as 51 it infects over 450 plant species (6-8) and its metabolism is poorly understood. It is a 52 soil borne pathogen that enters the plants through natural openings or wounds,...

Bacterial accumulation of poly­(3-hydroxybutyrate) [P­(3HB)] is a metabolic strategy often adopted to cope with challenging surroundings. Ralstonia solanacearum, a phytopathogen, seems to be an ideal candidate with inherent ability to accumulate this biodegradable polymer of high industrial relevance. This study is focused on investigating the metabolic networks that channel glucose into P­(3HB) using comparative genome analysis, 13C tracers, microscopy, gas chromatography-mass spectrometry (GC-MS), and proton nuclear magnetic resonance (1H NMR). Comparative genome annotation of 87 R. solanacearum strains confirmed the presence of a conserved P­(3HB) biosynthetic pathway genes in the chromosome. Parallel 13C glucose feeding ([1-13C], [1,2-13C]) analysis mapped the glucose oxidation to 3-hydroxybutyrate (3HB), the metabolic precursor of P­(3HB) via the Entner–Doudoroff pathway (ED pathway), potentially to meet the NADPH demands. Fluorescence microscopy, GC-MS, and 1H NMR analysis further confirmed the ability of R. solanacearum to accumulate P­(3HB) granules. In addition, it is demonstrated that the carbon/nitrogen (C/N) ratio influences the P­(3HB) yields, thereby highlighting the need to further optimize the bioprocessing parameters. This study provided key insights into the biosynthetic abilities of R. solanacearum as a promising P­(3HB) producer.

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