Assessing sequence heterogeneity in Chlorellaceae DNA barcode markers for phylogenetic inference.

Phylogenetic inference is an important approach that allows the recovery of the evolutionary history and the origin of the Chlorellaceae species. Despite the species' potential for biofuel feedstock production, their high phenotypic plasticity and similar morphological structures among the species have muddled the taxonomy and identification of the Chlorellaceae species. This study aimed to decipher Chlorellaceae DNA barcode marker heterogeneity by examining the sequence divergence and genomic properties of 18S rRNA, ITS (ITS1-5.8S rRNA-ITS2-28S rRNA), and rbcL from 655 orthologous sequences of 64 species across 31 genera in the Chlorellaceae family. The study assessed the distinct evolutionary properties of the DNA markers that may have caused the discordance between individual trees in the phylogenetic inference using the Robinson-Foulds distance and the Shimodaira-Hasegawa test. Our findings suggest that using the supermatrix approach improves the congruency between trees by reducing stochastic error and increasing the confidence of the inferred Chlorellaceae phylogenetic tree. This study also found that the phylogenies inferred through the supermatrix approach might not always be well supported by all markers. The study highlights that assessing sequence heterogeneity prior to the phylogenetic inference could allow the approach to accommodate sequence evolutionary properties and support species identification from the most congruent phylogeny, which can better represent the evolution of Chlorellaceae species.

Molecular and allergenic characterization of recombinant tropomyosin from mud crab Scylla olivacea.

BACKGROUND: Tropomyosin is a major allergen in crustaceans, including mud crab species, but its molecular and allergenic properties in Scylla olivacea are not well known. Thus, this study aimed to produce the recombinant tropomyosin protein from S. olivacea and subsequently investigate its IgE reactivity. METHODS AND RESULTS: The tropomyosin gene was cloned and expressed in the Escherichia coli system, followed by SDS-PAGE and immunoblotting test to identify the allergenic potential of the recombinant protein. The 855-base pair of tropomyosin gene produced was found to be 99.18% homologous to Scylla serrata. Its 284 amino acids matched the tropomyosin of crustaceans, arachnids, insects, and Klebsiella pneumoniae, ranging from 79.03 to 95.77%. The tropomyosin contained 89.44% alpha-helix folding with a tertiary structure of two-chain alpha-helical coiled-coil structures comprising a homodimer heptad chain. IPTG-induced histidine tagged-recombinant tropomyosin was purified at the size of 42 kDa and confirmed as tropomyosin using anti-tropomyosin monoclonal antibodies. The IgE binding of recombinant tropomyosin protein was reactive in 90.9% (20/22) of the sera from crab-allergic patients. CONCLUSIONS: This study has successfully produced an allergenic recombinant tropomyosin from S. olivacea. This recombinant tropomyosin may be used as a specific allergen for the diagnosis of allergy.

Inactivation of the catalytic subunit of cAMP-dependent protein kinase A causes delayed appressorium formation and reduced pathogenicity of Colletotrichum gloeosporioides.

The cyclic AMP- (cAMP-) dependent protein kinase A signaling pathway is one of the major signaling pathways responsible for regulation of the morphogenesis and pathogenesis of several pathogenic fungi. To evaluate the role of this pathway in the plant pathogenic fungus, Colletotrichum gloeosporioides, the gene encoding the catalytic subunit of cAMP-dependent protein kinase A, CgPKAC, was cloned, inactivated, and the mutant was analyzed. Analysis of the Cgpkac mutant generated via gene replacement showed that the mutants were able to form appressoria; however, their formation was delayed compared to the wild type. In addition, the mutant conidia underwent bipolar germination after appressoria formation, but no appressoria were generated from the second germ tube. The mutants also showed reduced ability to adhere to a hydrophobic surface and to degrade lipids localized in the appressoria. Based on the number of lesions produced during a pathogenicity test, the mutant's ability to cause disease in healthy mango fruits was reduced, which may be due to failure to penetrate into the fruit. These findings indicate that cAMP-dependent protein kinase A has an important role in regulating morphogenesis and is required for pathogenicity of C. gloeosporioides.