Functional Characterization of a Melon Alcohol Acyl-transferase Gene Family Involved in the Biosynthesis of Ester Volatiles. Identification of the Crucial Role of a Threonine Residue for Enzyme Activity*

ABSTRACT Human diseases caused by species of Aeromonas have been classified into two major groups: septicemia and gastroenteritis. In this study, we reported the molecular and functional characterization of a new virulence factor, ToxR-regulated lipoprotein, or TagA, from a diarrheal isolate, SSU, of Aeromonas hydrophila. The tagA gene of A. hydrophila exhibited 60% identity with that of a recently identified stcE gene from Escherichia coli O157:H7, which encoded a protein (StcE) that provided serum resistance to the bacterium and prevented erythrocyte lysis by controlling classical pathway of complement activation by cleaving the complement C1-esterase inhibitor (C1-INH). We purified A. hydrophila TagA as a histidine-tagged fusion protein (rTagA) from E. coli DE3 strain using a T7 promoter-based pET30 expression vector and nickel affinity column chromatography. rTagA cleaved C1-INH in a time-dependent manner. The tagA isogenic mutant of A. hydrophila, unlike its corresponding wild-type (WT) or the complemented strain, was unable to cleave C1-INH, which is required to potentiate the C1-INH-mediated lysis of host and bacterial cells. We indeed demonstrated colocalization of C1-INH and TagA on the bacterial surface by confocal fluorescence microscopy, which ultimately resulted in increased serum resistance of the WT bacterium. Likewise, we delineated the role of TagA in contributing to the enhanced ability of C1-INH to inhibit the classical complement-mediated lysis of erythrocytes. Importantly, we provided evidence that the tagA mutant was significantly less virulent in a mouse model of infection (60%) than the WT bacterium at two 50% lethal doses, which resulted in 100% mortality within 48 h. Taken together, our data provided new information on the role of TagA as a virulence factor in bacterial pathogenesis. This is the first report of TagA characterization from any species of Aeromonas.

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Functional Characterization of Spliceosomal Introns and Identification of U2, U4, and U5 snRNAs in the Deep-Branching Eukaryote Entamoeba histolytica

Eukaryotic Cell ◽

10.1128/ec.00059-07 ◽

2007 ◽

Vol 6 (6) ◽

pp. 940-948 ◽

Cited By ~ 16

Author(s):

Carrie A. Davis ◽

Michael P. S. Brown ◽

Upinder Singh

Keyword(s):

Entamoeba Histolytica ◽

Splice Site ◽

Expressed Sequence Tag ◽

Functional Characterization ◽

Molecular Evidence ◽

Spliceosomal Introns ◽

Accurate Expression ◽

Eukaryotic Organisms

ABSTRACT Pre-mRNA splicing is essential to ensure accurate expression of many genes in eukaryotic organisms. In Entamoeba histolytica, a deep-branching eukaryote, approximately 30% of the annotated genes are predicted to contain introns; however, the accuracy of these predictions has not been tested. In this study, we mined an expressed sequence tag (EST) library representing 7% of amoebic genes and found evidence supporting splicing of 60% of the testable intron predictions, the majority of which contain a GUUUGU 5′ splice site and a UAG 3′ splice site. Additionally, we identified several splice site misannotations, evidence for the existence of 30 novel introns in previously annotated genes, and identified novel genes through uncovering their spliced ESTs. Finally, we provided molecular evidence for the E. histolytica U2, U4, and U5 snRNAs. These data lay the foundation for further dissection of the role of RNA processing in E. histolytica gene expression.

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Genome-wide identification and functional characterization of Camelina sativa WRKY gene family in response to abiotic stress

10.21203/rs.3.rs-36113/v3 ◽

2020 ◽

Author(s):

Yanan Song ◽

Hongli Cui ◽

Ying Shi ◽

Jinai Xue ◽

Chunli Ji ◽

...

Keyword(s):

Gene Family ◽

Stress Responses ◽

Stress Resistance ◽

Segmental Duplication ◽

Functional Characterization ◽

High Stress ◽

Camelina Sativa ◽

Genome Wide ◽

Large Expansion

Abstract Background: WRKY transcription factors are a superfamily of regulators involved in diverse biological processes and stress responses in plants. However, knowledge is limited for WRKY family in camelina (Camelina sativa), an important Brassicaceae oil crop with strong tolerance against various stresses. Here, genome-wide characterization of WRKY proteins is performed to examine their gene-structures, phylogenetics, expressions, conserved motif organizations, and functional annotation to identify candidate WRKYs mediating regulation of stress resistance in camelina.Results: Total of 242 CsWRKY proteins encoded by 224 gene loci distributed uneven on chromosomes were identified, and classified into three groups via phylogenetic analysis according to their WRKY domains and zinc finger motifs. 15 CsWRKY gene loci generated 33 spliced variants. Orthologous WRKY gene pairs were identified, with 173 pairs in C. sativa and Arabidopsis genomes as well as 282 pairs for C. sativa and B. napus, respectively. 137 segmental duplication events were observed but no tandem duplication in camelina genome. Ten major conserved motifs were examined, with WRKYGQK as the most conserved and several variants existed in many CsWRKYs. Expression analysis revealed that half more CsWRKY genes were expressed constitutively, and a set of them had a tissue-specific expression. Notably, 11 CsWRKY genes exhibited significantly expression changes in plant seedlings under cold, salt, and drought stress, respectively, having preferentially inducible expression pattern in response to the stress.Conclusions: The present described a detail analysis of CsWRKY gen family and their expression profiled in twelve tissues and under several stress conditions. Segmental duplication is the major force for large expansion of this gene family, and a strong purifying pressure happened for CsWRKY proteins evolutionally. CsWRKY proteins play important roles for plant development, with differential functions in different tissues. Exceptionally, eleven CsWRKYs, particularly five alternative spliced isoforms were found to be the key players possibly in mediating plant response to various stresses. Overall, our results provide a foundation for understanding roles of CsWRKYs and the precise mechanism through which CsWRKYs regulate high stress resistance to stress as well as development of stress tolerance cultivars for Cruciferae crops.

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Identification of an isoflavonoid transporter required for the nodule establishment of the Rhizobium-Fabaceae symbiotic interaction

10.1101/2021.07.30.452494 ◽

2021 ◽

Author(s):

Wanda Biala-Leonhard ◽

Laura Zanin ◽

Stefano Gottardi ◽

Rita de Brito Francisco ◽

Silvia Venuti ◽

...

Keyword(s):

Functional Characterization ◽

Crop Productivity ◽

Signaling Cascades ◽

Nutritional Deficiencies ◽

White Lupin ◽

Symbiotic Interaction ◽

Phosphate Availability ◽

Rhizobial Symbiosis

Nitrogen (N) as well as Phosphorus (P) are key nutrients determining crop productivity. Legumes have developed strategies to overcome nutrient limitation by e.g., forming a symbiotic relationship with N-fixing rhizobia and the release of P-mobilizing exudates and are thus able to grow without supply of N or P fertilizers. The legume-rhizobial symbiosis starts with root release of isoflavonoids, that act as signaling molecules perceived by compatible bacteria. Subsequently, bacteria release nod factors, which induce signaling cascades allowing the formation of functional N-fixing nodules. We report here the identification and functional characterization of a plasma membrane-localized MATE-type transporter (LaMATE2) involved in the release of genistein from white lupin roots. The LaMATE2 expression in the root is upregulated under N deficiency as well as low phosphate availability, two nutritional deficiencies that induce the release of this isoflavonoid. LaMATE2 silencing reduced genistein efflux and even more the formation of symbiotic nodules, supporting the crucial role of LaMATE2 in isoflavonoid release and nodulation. Furthermore, silencing of LaMATE2 limited the P-solubilization activity of lupin root exudates. Transport assays in yeast vesicles demonstrated that LaMATE2 acts as a proton-driven isoflavonoid transporter.

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