Sarosh Bejai

Complete genome sequence of a plant associated bacterium Bacillus amyloliquefaciens subsp. plantarum UCMB5033

Bacillus amyloliquefaciens subsp. plantarum UCMB5033 is of special interest for its ability to promote host plant growth through production of stimulating compounds and suppression of soil borne pathogens by synthesizing antibacterial and antifungal metabolites or priming plant defense as induced systemic resistance. The genome of B. amyloliquefaciens UCMB5033 comprises a 4,071,167 bp long circular chromosome that consists of 3,912 protein-coding genes, 86 tRNA genes and 10 rRNA operons.

Myrosinases from root and leaves of Arabidopsis thaliana have different catalytic properties

Myrosinases (EC 3.2.1.147) are beta-thioglucoside glucosidases present in Brassicaceae plants. These enzymes serve to protect plants against pathogens and insect pests by initiating breakdown of the secondary metabolites glucosinolates into toxic products. Several forms of myrosinases are present in plants but the properties and role of different isoenzymes are not well understood. The dicot plant model organism Arabidopsis thaliana seems to contain six myrosinase genes (TGG1-TGG6). In order to compare the different myrosinases, cDNAs corresponding to TGG1 from leaves and TGG4 and TGG5 from roots were cloned and overexpressed in Pichia pastoris. The His-tagged recombinant proteins were purified using affinity chromatography and the preparations were homogenous according to SDS-PAGE analysis. Myrosinase activity was confirmed for all forms and compared with respect to catalytic activity towards the allyl-glucosinolate sinigrin. There was a 22-fold difference in basal activity among the myrosinases. The enzymes were active in a broad pH range, are rather thermostable and active in a wide range of salt concentrations but sensitive to high salt concentrations. The myrosinases showed different activation-inhibition responses towards ascorbic acid with maximal activity around 0.7-1 mM. No activity was registered towards desulphosinigrin and this compound did not inhibit myrosinase activity towards sinigrin. All myrosinases also displayed O-beta-glucosidase activity, although with lower efficiency compared to the myrosinase activity. The differences in catalytic properties among myrosinase isozymes for function in planta are discussed.

Genome Analysis of Bacillus amyloliquefaciens Subsp. plantarum UCMB5113: A Rhizobacterium That Improves Plant Growth and Stress Management

The Bacillus amyloliquefaciens subsp. plantarum strain UCMB5113 is a Gram-positive rhizobacterium that can colonize plant roots and stimulate plant growth and defense based on unknown mechanisms. This reinforcement of plants may provide protection to various forms of biotic and abiotic stress. To determine the genetic traits involved in the mechanism of plant-bacteria association, the genome sequence of UCMB5113 was obtained by assembling paired-end Illumina reads. The assembled chromosome of 3,889,532 bp was predicted to encode 3,656 proteins. Genes that potentially contribute to plant growth promotion such as indole-3-acetic acid (IAA) biosynthesis, acetoin synthesis and siderophore production were identified. Moreover, annotation identified putative genes responsible for non-ribosomal synthesis of secondary metabolites and genes supporting environment fitness of UCMB5113 including drug and metal resistance. A large number of genes encoding a diverse set of secretory proteins, enzymes of primary and secondary metabolism and carbohydrate active enzymes were found which reflect a high capacity to degrade various rhizosphere macromolecules. Additionally, many predicted membrane transporters provides the bacterium with efficient uptake capabilities of several nutrients. Although, UCMB5113 has the possibility to produce antibiotics and biosurfactants, the protective effect of plants to pathogens seems to be indirect and due to priming of plant induced systemic resistance. The availability of the genome enables identification of genes and their function underpinning beneficial interactions of UCMB5113 with plants.

Improved heat stress tolerance of wheat seedlings by bacterial seed treatment

AimTo investigate if rhizosphere bacteria can improve heat tolerance of wheat.MethodsWheat (Triticum aestivum) seeds of the cultivars Olivin and Sids1 were treated with Bacillus amyloliquefaciens UCMB5113 or Azospirillum brasilense NO40 and young seedlings tested for management of short term heat stress.ResultsBacterial treatment improved heat stress management of wheat. Olivin showed higher heat tolerance than Sids1 both with non-inoculated and inoculated seeds. Heat increased transcript levels of several stress related genes in the leaves, while expression was lower in inoculated plants but elevated compared with the control. Enzymes of the ascorbate-glutathione redox cycle were activated in leaves after heat challenge but showed a lower response in inoculated plants. Metabolite profiling distinguished different treatments dependent on analysis technique with respect to primary and secondary metabolites. Analysis of some plant stress regulatory genes showed that bacterial treatment increased transcript levels while effects of heat treatment varied.ConclusionsThe improvement of heat tolerance by bacteria seems associated with reduced generation of reactive oxygen species (and consequently less cell damage), small changes in the metabolome while preactivation of certain heat shock transcription factors seems important. Seed inoculation with beneficial bacteria seems a promising strategy to improve heat tolerance of wheat.

Multiple effects of Bacillus amyloliquefaciens volatile compounds: plant growth promotion and growth inhibition of phytopathogens

Biotic interactions through volatile organic compounds (VOC) are frequent in nature. This investigation aimed to study the role of ITALIC! BacillusVOC for the beneficial effects on plants observed as improved growth and pathogen control. Four ITALIC! Bacillus amyloliquefacienssubsp. ITALIC! plantarumstrains were screened for VOC effects on ITALIC! Arabidopsis thalianaCol-0 seedlings and ITALIC! Brassicafungal phytopathogens. VOC from all four ITALIC! Bacillusstrains could promote growth of ITALIC! Arabidopsisplants resulting in increased shoot biomass but the effects were dependent on the growth medium. Dose response studies with UCMB5113 on MS agar with or without root exudates showed significant plant growth promotion even at low levels of bacteria. ITALIC! BacillusVOC antagonized growth of several fungal pathogens ITALIC! in vitro However, the plant growth promotion efficacy and fungal inhibition potency varied among the ITALIC! Bacillusstrains. VOC inhibition of several phytopathogens indicated efficient microbial antagonism supporting high rhizosphere competence of the ITALIC! Bacillusstrains. GC-MS analysis identified several VOC structures where the profiles differed depending on the growth medium. The ability of ITALIC! Bacillusstrains to produce both volatile and soluble compounds for plant growth promotion and disease biocontrol provides examples of rhizosphere microbes as an important ecosystem service with high potential to support sustainable crop production.

RabGAP22 Is Required for Defense to the Vascular Pathogen Verticillium longisporum and Contributes to Stomata Immunity

Verticillium longisporum is a soil-borne pathogen with a preference for plants within the family Brassicaceae. Following invasion of the roots, the fungus proliferates in the plant vascular system leading to stunted plant growth, chlorosis and premature senescence. RabGTPases have been demonstrated to play a crucial role in regulating multiple responses in plants. Here, we report on the identification and characterization of the Rab GTPase-activating protein RabGAP22 gene from Arabidopsis, as an activator of multiple components in the immune responses to V. longisporum. RabGAP22Pro:GUS transgenic lines showed GUS expression predominantly in root meristems, vascular tissues and stomata, whereas the RabGAP22 protein localized in the nucleus. Reduced RabGAP22 transcript levels in mutants of the brassinolide (BL) signaling gene BRI1-ASSOCIATED RECEPTOR KINASE 1, together with a reduction of fungal proliferation following BL pretreatment, suggested RabGAP22 to be involved in BL-mediated responses. Pull-down assays revealed SERINE:GLYOXYLATE AMINOTRANSFERASE (AGT1) as an interacting partner during V. longisporum infection and bimolecular fluorescence complementation (BiFC) showed the RabGAP22-AGT1 protein complex to be localized in the peroxisomes. Further, fungal-induced RabGAP22 expression was found to be associated with elevated endogenous levels of the plant hormones jasmonic acid (JA) and abscisic acid (ABA). An inadequate ABA response in rabgap22-1 mutants, coupled with a stomata-localized expression of RabGAP22 and impairment of guard cell closure in response to V. longisporum and Pseudomonas syringae, suggest that RabGAP22 has multiple roles in innate immunity.

Analysis of plant growth-promoting properties of Bacillus amyloliquefaciens UCMB5113 using Arabidopsis thaliana as host plant.

AbstractMain conclusionThis study showed thatBacillusamyloliquefaciensUCMB5113 colonizingArabidopsisroots changed root structure and promoted growth implying the usability of this strain as a novel tool to support sustainable crop production. Root architecture plays a crucial role for plants to ensure uptake of water, minerals and nutrients and to provide anchorage in the soil. The root is a dynamic structure with plastic growth and branching depending on the continuous integration of internal and environmental factors. The rhizosphere contains a complex microbiota, where some microbes can colonize plant roots and support growth and stress tolerance. Here, we report that the rhizobacterium Bacillus amyloliquefaciens subsp. plantarum UCMB5113 stimulated the growth of Arabidopsisthaliana Col-0 by increased lateral root outgrowth and elongation and root-hair formation, although primary root elongation was inhibited. In addition, the growth of the above ground tissues was stimulated by UCMB5113. Specific hormone reporter gene lines were tested which suggested a role for at least auxin and cytokinin signaling during rhizobacterial modulation of Arabidopsis root architecture. UCMB5113 produced cytokinins and indole-3-acetic acid, and the formation of the latter was stimulated by root exudates and tryptophan. The plant growth promotion effect by UCMB5113 did not appear to depend on jasmonic acid in contrast to the disease suppression effect in plants. UCMB5113 exudates inhibited primary root growth, while a semi-purified lipopeptide fraction did not and resulted in the overall growth promotion indicating an interplay of many different bacterial compounds that affect the root growth of the host plant. This study illustrates that beneficial microbes interact with plants in root development via classic and novel signals.

Genome Sequence of a Plant-Associated Bacterium, Bacillus amyloliquefaciens Strain UCMB5036.

ABSTRACT We announce here the genome sequence of Bacillus amyloliquefaciens strain UCMB5036, a plant growth-promoting bacterium isolated from a cotton plant. Its genome contains gene clusters involved in nonribosomal synthesis of secondary metabolites known for their antimicrobial activities. The availability of this genome will provide novel insights into plant-bacterium–associated activities.

ML3: a novel regulator of herbivory-induced responses in Arabidopsis thaliana

ML (MD2-related lipid recognition) proteins are known to enhance innate immune responses in mammals. This study reports the analysis of the putative ML gene family in Arabidopsis thaliana and suggests a role for the ML3 gene in herbivory-associated responses in plants. Feeding by larvae of the Lepidopteran generalist herbivore Spodoptera littoralis and larvae of the specialist herbivore Plutella xylostella activated ML3 transcription in leaf tissues. ML3 loss-of-function Arabidopsis plants were compromised in the upregulation of herbivory-induced genes and displayed a semi-dwarf phenotype. Herbivory bioassays showed that larvae of S. littoralis fed on ml3 mutant plants gained more weight compared to larvae fed on wild-type plants while larvae of P. xylostella did not show any significant difference. Virus-induced gene silencing of ML3 expression in plants compromised in jasmonic acid (JA) and salicylic acid (SA) signalling revealed a complex role of ML3 in JA/defence signalling affecting both JA- and SA-dependent responses. The data suggest that ML3 is involved in herbivory-mediated responses in Arabidopsis and that it has a potential role in herbivory-associated molecular pattern recognition.

ML3 Is a NEDD8- and Ubiquitin-Modified Protein

A previously uncharacterized lipid-binding protein is modified by the ubiquitin-like protein NEDD8 and by ubiquitin. NEDD8 (NEURAL PRECURSOR CELL-EXPRESSED, DEVELOPMENTALLY DOWN-REGULATED PROTEIN8) is an evolutionarily conserved 8-kD protein that is closely related to ubiquitin and that can be conjugated like ubiquitin to specific lysine residues of target proteins in eukaryotes. In contrast to ubiquitin, for which a broad range of substrate proteins are known, only a very limited number of NEDD8 target proteins have been identified to date. Best understood, and also evolutionarily conserved, is the NEDD8 modification (neddylation) of cullins, core subunits of the cullin-RING-type E3 ubiquitin ligases that promote the polyubiquitylation of degradation targets in eukaryotes. Here, we show that Myeloid differentiation factor-2-related lipid-recognition domain protein ML3 is an NEDD8- as well as ubiquitin-modified protein in Arabidopsis (Arabidopsis thaliana) and examine the functional role of ML3 in the plant cell. Our analysis indicates that ML3 resides in the vacuole as well as in endoplasmic reticulum (ER) bodies. ER bodies are Brassicales-specific ER-derived organelles and, similar to other ER body proteins, ML3 orthologs can only be identified in this order of flowering plants. ML3 gene expression is promoted by wounding as well as by the phytohormone jasmonic acid and repressed by ethylene, signals that are known to induce and repress ER body formation, respectively. Furthermore, ML3 protein abundance is dependent on NAI1, a master regulator of ER body formation in Arabidopsis. The regulation of ML3 expression and the localization of ML3 in ER bodies and the vacuole is in agreement with a demonstrated importance of ML3 in the defense to herbivore attack. Here, we extend the spectrum of ML3 biological functions by demonstrating a role in the response to microbial pathogens.