scholarly journals Identification and Functional Analysis of NLP-Encoding Genes from the Postharvest Pathogen Penicillium expansum

2019 ◽  
Vol 7 (6) ◽  
pp. 175 ◽  
Author(s):  
Elena Levin ◽  
Ginat Raphael ◽  
Jing Ma ◽  
Ana-Rosa Ballester ◽  
Oleg Feygenberg ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Nicotiana Benthamiana ◽  
Liquid Culture ◽  
Functional Characterization ◽  
Penicillium Expansum ◽  
Lesion Development ◽  
Fruit Tissue ◽  
Reduced Rate ◽  
Encoding Genes

Penicillium expansum is a major postharvest pathogen that infects different fruits, mainly through injuries inflicted during harvest or subsequent handling after harvest. Several effectors were suggested to mediate pathogenicity of P. expansum in fruit tissue. Among these effectors Nep1-like proteins (NLPs), produced by various microorganisms with different lifestyles, are known for their ability to induce necrosis in dicot plants and were shown to be involved in virulence of several plant-related pathogens. This study was aimed at the identification and functional characterization of two NLP genes found in the genome of P. expansum. The genes were designated Penlp1 and Penlp2 and were found to code type1 and type3 NLP respectively. Necrosis-inducing activity of the two proteins was demonstrated by transient expression in Nicotiana benthamiana leaves. While Penlp1 expression was induced during apple infection and in liquid culture, the highest level of Penlp2 expression was found in ungerminated spores. Deletion of Penlp1, but not Penlp2, resulted in reduced virulence on apples manifested by reduced rate of lesion development (disease severity).

scholarly journals Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 524
Author(s):  
Bingqi Wu ◽  
Zhiting Chen ◽  
Xiaohui Xu ◽  
Ronghua Chen ◽  
Siwei Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Functional Characterization ◽  
Transient Gene Expression ◽  
High Mobility ◽  
Gene Expression System

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

scholarly journals Structural and functional analysis of two di-domain aromatase/cyclases from type II polyketide synthases

2015 ◽  
Vol 112 (50) ◽  
pp. E6844-E6851 ◽  
Author(s):  
Grace Caldara-Festin ◽  
David R. Jackson ◽  
Jesus F. Barajas ◽  
Timothy R. Valentic ◽  
Avinash B. Patel ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Functional Role ◽  
Functional Characterization ◽  
Polyketide Synthases ◽  
Type Ii ◽  
Aromatic Polyketides ◽  
Linear Poly ◽  
Bacterial Type

Aromatic polyketides make up a large class of natural products with diverse bioactivity. During biosynthesis, linear poly-β-ketone intermediates are regiospecifically cyclized, yielding molecules with defined cyclization patterns that are crucial for polyketide bioactivity. The aromatase/cyclases (ARO/CYCs) are responsible for regiospecific cyclization of bacterial polyketides. The two most common cyclization patterns are C7–C12 and C9–C14 cyclizations. We have previously characterized three monodomain ARO/CYCs: ZhuI, TcmN, and WhiE. The last remaining uncharacterized class of ARO/CYCs is the di-domain ARO/CYCs, which catalyze C7–C12 cyclization and/or aromatization. Di-domain ARO/CYCs can further be separated into two subclasses: “nonreducing” ARO/CYCs, which act on nonreduced poly-β-ketones, and “reducing” ARO/CYCs, which act on cyclized C9 reduced poly-β-ketones. For years, the functional role of each domain in cyclization and aromatization for di-domain ARO/CYCs has remained a mystery. Here we present what is to our knowledge the first structural and functional analysis, along with an in-depth comparison, of the nonreducing (StfQ) and reducing (BexL) di-domain ARO/CYCs. This work completes the structural and functional characterization of mono- and di-domain ARO/CYCs in bacterial type II polyketide synthases and lays the groundwork for engineered biosynthesis of new bioactive polyketides.

scholarly journals Mutagenesis and Functional Characterization of the Four Domains of GlnD, a Bifunctional Nitrogen Sensor Protein

2010 ◽  
Vol 192 (11) ◽  
pp. 2711-2721 ◽  
Author(s):  
Yaoping Zhang ◽  
Edward L. Pohlmann ◽  
Jose Serate ◽  
Mary C. Conrad ◽  
Gary P. Roberts
Keyword(s):  
Escherichia Coli ◽  
Functional Analysis ◽  
Nitrogen Assimilation ◽  
Rhodospirillum Rubrum ◽  
Functional Characterization ◽  
Enteric Bacteria ◽  
Truncated Protein ◽  
Primary Sensor ◽  
Sensor Protein

ABSTRACT GlnD is a bifunctional uridylyltransferase/uridylyl-removing enzyme (UTase/UR) and is believed to be the primary sensor of nitrogen status in the cell by sensing the level of glutamine in enteric bacteria. It plays an important role in nitrogen assimilation and metabolism by reversibly regulating the modification of PII protein; PII in turn regulates a variety of other proteins. GlnD appears to have four distinct domains: an N-terminal nucleotidyltransferase (NT) domain; a central HD domain, named after conserved histidine and aspartate residues; and two C-terminal ACT domains, named after three of the allosterically regulated enzymes in which this domain is found. Here we report the functional analysis of these domains of GlnD from Escherichia coli and Rhodospirillum rubrum. We confirm the assignment of UTase activity to the NT domain and show that the UR activity is a property specifically of the HD domain: substitutions in this domain eliminated UR activity, and a truncated protein lacking the NT domain displayed UR activity. The deletion of C-terminal ACT domains had little effect on UR activity itself but eliminated the ability of glutamine to stimulate that activity, suggesting a role for glutamine sensing by these domains. The deletion of C-terminal ACT domains also dramatically decreased UTase activity under all conditions tested, but some of these effects are due to the competition of UTase activity with unregulated UR activity in these variants.

scholarly journals Functional Characterization of Burkholderia pseudomallei Trimeric Autotransporters

2013 ◽  
Vol 81 (8) ◽  
pp. 2788-2799 ◽  
Author(s):  
Cristine G. Campos ◽  
Matthew S. Byrd ◽  
Peggy A. Cotter
Keyword(s):  
Burkholderia Pseudomallei ◽  
Functional Characterization ◽  
A549 Cells ◽  
Content Type ◽  
Tier 1 ◽  
Type V ◽  
Mouse Model Of Infection ◽  
Encoding Genes

ABSTRACTBurkholderia pseudomalleiis a tier 1 select agent and the causative agent of melioidosis, a severe and often fatal disease with symptoms ranging from acute pneumonia and septic shock to a chronic infection characterized by abscess formation in the lungs, liver, and spleen. Autotransporters (ATs) are exoproteins belonging to the type V secretion system family, with many playing roles in pathogenesis. The genome ofB. pseudomalleistrain 1026b encodes nine putative trimeric AT proteins, of which only four have been described. Using a bioinformatic approach, we annotated putative domains within each trimeric AT protein, excluding the well-studied BimA protein, and found short repeated sequences unique toBurkholderiaspecies, as well as an unexpectedly large proportion of ATs with extended signal peptide regions (ESPRs). To characterize the role of trimeric ATs in pathogenesis, we constructed disruption or deletion mutations in each of eight AT-encoding genes and evaluated the resulting strains for adherence to, invasion of, and plaque formation in A549 cells. The majority of the ATs (and/or the proteins encoded downstream) contributed to adherence to and efficient invasion of A549 cells. Using a BALB/c mouse model of infection, we determined the contributions of each AT to bacterial burdens in the lungs, liver, and spleen. At 48 h postinoculation, only one strain, Bp340::pDbpaC, demonstrated a defect in dissemination and/or survival in the liver, indicating that BpaC is required for wild-type virulence in this model.

TOWARDS THE FUNCTIONAL CHARACTERIZATION OF THE CLEMENTINE ASP-RICH PROTEIN ENCODING GENES, CANDIDATES FOR REGULATING GAMETOPHYTIC SELF-INCOMPATIBILITY

2015 ◽  
pp. 599-604 ◽  
Author(s):  
Marco Caruso ◽  
Luca Lo Cicero ◽  
Gaetano Distefano ◽  
Stefano La Malfa ◽  
Angela Roberta Lo Piero ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Self Incompatibility ◽  
Protein Encoding ◽  
Encoding Genes

scholarly journals Establishing a System for Functional Characterization of Full-Length cDNAs of Camellia sinensis

2019 ◽  
Vol 20 (23) ◽  
pp. 5929
Author(s):  
Lin ◽  
Cai ◽  
Du ◽  
Zhang ◽  
Xu ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Heterologous Expression ◽  
Cdna Library ◽  
Camellia Sinensis ◽  
Functional Characterization ◽  
Full Length ◽  
Rare Gene ◽  
Cdna Collection ◽  
Reference Genomes

: Tea (Camellia sinensis) is enriched with bioactive secondary metabolites, and is one of the most popular nonalcoholic beverages globally. Two tea reference genomes have been reported; however, the functional analysis of tea genes has lagged, mainly due to tea’s recalcitrance to genetic transformation and the absence of alternative high throughput heterologous expression systems. A full-length cDNA collection with a streamlined cloning system is needed in this economically important woody crop species. RNAs were isolated from nine different vegetative tea tissues, pooled, then used to construct a normalized full-length cDNA library. The titer of unamplified and amplified cDNA library was 6.89 × 106 and 1.8 × 1010 cfu/mL, respectively; the library recombinant rate was 87.2%. Preliminary characterization demonstrated that this collection can complement existing tea reference genomes and facilitate rare gene discovery. In addition, to streamline tea cDNA cloning and functional analysis, a binary vector (pBIG2113SF) was reengineered, seven tea cDNAs isolated from this library were successfully cloned into this vector, then transformed into Arabidopsis. One FL-cDNA, which encodes a putative P1B-type ATPase 5 (CsHMA5), was characterized further as a proof of concept. We demonstrated that overexpression of CsHMA5 in Arabidopsis resulted in copper hyposensitivity. Thus, our data demonstrated that this represents an efficient system for rare gene discovery and functional characterization of tea genes. The integration of a tea FL-cDNA collection with efficient cloning and a heterologous expression system would facilitate functional annotation and characterization of tea genes.

scholarly journals Functional Characterization of a Dendrobium officinale Geraniol Synthase DoGES1 Involved in Floral Scent Formation

2020 ◽  
Vol 21 (19) ◽  
pp. 7005
Author(s):  
Conghui Zhao ◽  
Zhenming Yu ◽  
Jaime A. Teixeira da Silva ◽  
Chunmei He ◽  
Haobin Wang ◽  
...  
Keyword(s):  
Nicotiana Benthamiana ◽  
Floral Scent ◽  
Functional Characterization ◽  
Dendrobium Officinale ◽  
Transcript Levels ◽  
Geraniol Synthase ◽  
Medicinal Orchid

Floral scent is a key ornamental trait that determines the quality and commercial value of orchids. Geraniol, an important volatile monoterpene in orchids that attracts pollinators, is also involved in responses to stresses but the geraniol synthase (GES) responsible for its synthesis in the medicinal orchid Dendrobium officinale has not yet been identified. In this study, three potential geraniol synthases were mined from the D. officinale genome. DoGES1, which was localized in chloroplasts, was characterized as a geraniol synthase. DoGES1 was highly expressed in flowers, especially in petals. DoGES1 transcript levels were high in the budding stage of D. officinale flowers at 11:00 a.m. DoGES1 catalyzed geraniol in vitro, and transient expression of DoGES1 in Nicotiana benthamiana leaves resulted in the accumulation of geraniol in vivo. These findings on DoGES1 advance our understanding of geraniol biosynthesis in orchids, and lay the basis for genetic modification of floral scent in D. officinale or in other ornamental orchids.

Sign in / Sign up

Export Citation Format

Share Document