Characterization of the Chitinase Gene Family in Mulberry (Morus notabilis) and MnChi18 Involved in Resistance to Botrytis cinerea

Chitinase is a hydrolase that uses chitin as a substrate. It plays an important role in plant resistance to fungal pathogens by degrading chitin. Here, we conducted bioinformatics analysis and transcriptome data analysis of the mulberry (Morus notabilis) chitinase gene family to determine its role in the resistance to Botrytis cinerea. A total of 26 chitinase genes were identified, belonging to the GH18 and GH19 families. Among them, six chitinase genes were differentially expressed under the infection of B. cinerea. MnChi18, which significantly responded to B. cinerea, was heterologously expressed in Arabidopsis (Arabidopsis thaliana). The resistance of MnChi18 transgenic Arabidopsis to B. cinerea was significantly enhanced, and after inoculation with B. cinerea, the activity of catalase (CAT) increased and the content of malondialdehyde (MDA) decreased. This shows that overexpression of MnChi18 can protect cells from damage. In addition, our study also indicated that MnChi18 may be involved in B. cinerea resistance through other resistance-related genes. This study provides an important basis for further understanding the function of mulberry chitinase.

The Interactions among Isolates of Lactiplantibacillus plantarum and Dairy Yeast Contaminants: Towards Biocontrol Applications

Yeast diversity in the cheese manufacturing process and in the cheeses themselves includes indispensable species for the production of specific cheeses and undesired species that cause cheese defects and spoilage. The control of yeast contaminants is problematic due to limitations in sanitation methods and chemicals used in the food industry. The utilisation of lactic acid bacteria and their antifungal products is intensively studied. Lactiplantibacillus plantarum is one of the most frequently studied species producing a wide spectrum of bioactive by-products. In the present study, twenty strains of L. plantarum from four sources were tested against 25 species of yeast isolated from cheeses, brines, and dairy environments. The functional traits of L. plantarum strains, such as the presence of class 2a bacteriocin and chitinase genes and in vitro production of organic acids, were evaluated. The extracellular production of bioactive peptides and proteins was tested using proteomic methods. Antifungal activity against yeast was screened using in vitro tests. Testing of antifungal activity on artificial media and reconstituted milk showed significant variability within the strains of L. plantarum and its group of origin. Strains from sourdoughs (CCDM 3018, K19-3) and raw cheese (L12, L24, L32) strongly inhibited the highest number of yeast strains on medium with reconstituted milk. These strains showed a consistent spectrum of genes belonging to class 2a bacteriocins, the gene of chitinase and its extracellular product 9 LACO Chitin-binding protein. Strain CCDM 3018 with the spectrum of class 2a bacteriocin gene, chitinase and significant production of lactic acid in all media performed significant antifungal effects in artificial and reconstituted milk-based media.

Enhanced Resistance To Fungal Pathogens in Transgenic Peanut (Arachis Hypogaea L.) Cultivar L14 by Overexpression of Gene encoding Chitinase 42 kDa from Trichoderma Asperellum SH16

This study reports the expression of 42 kDa chitinase genes from Trichoderma asperellum SH16 in peanut (Arachis hypogaea) roots under the regulation of tissue-specific Asy promoter through Agrobacterium tumefaciens-mediated transformation. The 42 kDa chitinase genes, including one wild-type sequence (Chi42) and two synthetic sequences (syncodChi42-1 and syncodChi42-2) which were optimized for codon usage for plant expression, were incorporated into the peanut genome and successfully expressed in their roots. The investigation revealed that the enzyme chitinase from two synthetic genes had higher activity than that from the wild-type gene, about 901 U/mg (140 U/mL) and 1124 U/mg (197 U/mL) vs about 508 U/mg (87 U/mL). Transgenic peanut roots also exhibited extracellular chitinase activity which was driven by signal peptide of rice amylase 3D gene against the pathogenic fungus Sclerotium rolfsii under in vitro conditions. The higher chitinase activity of two synthetic genes in peanut roots promises potential applications in the field of transgenic crops against phytopathogenic fungi.

A Comparative Transcriptome Analysis Reveals New Insights into Pre-Harvest Sprouting (PHS) in Wheat

Background: pre-harvest sprouting (PHS) is a significant cause of yield loss in cereal crops, and is an important topic of study for the improvement of wheat quality. Many studies have focused on PHS in wheat during the last 10 years, especially on the involvement of abscisic acid (ABA) in PHS, however, a lot remained unknown about this topic.Results: In this study, a PHS resistant line was isolated from an ethylmethane sulfonate (EMS) mutant population derived from the wheat cultivar ‘Long 13-3778’, namely ‘LQ18’. The mutant line LQ18 showed highly significant resistance to PHS compared with the wild-type. Transcriptome sequencing was conducted to determine the differences between the LQ18 mutant and the wild-type at the level of gene expression. The results showed no conclusive evidence that the ABA biosynthesis and signaling pathways contribute to the differences in PHS between the mutant and the wild-type, and some genes and their alleles associated with PHS tolerance showed differential expression between the mutant and wild-type lines. The most interesting result of this study was that the expression levels of the chitinase family genes showed significant differences between the mutant and the wild-type as determined by GO enrichment analysis, and a subsequent analysis of differential expression profiling of the chitinase genes led to the same conclusion.Conclusions: Transcriptomic analysis in this study have revealed the global transcriptome profiles of the PHS sensitive wheat cultivar ‘Long 13-3778’ and its PHS resistant mutants. Furthermore, this study has proposed a possible explanation of the connection between PHS and the chitinase family for the first time, which added to our understanding of PHS and seed dormancy in common wheat.

Screening of chitinase-producing rhizosphere actinomycetes and their genetic diversity

Wahyudi AT, Fitriansyah NG, Amri MF, Priyanto JA, Nawangsih AA. 2021. Screening of chitinase-producing rhizosphere actinomycetes and their genetic diversity. Biodiversitas 22: 4186-4192. Soil microbial community, especially rhizosphere actinomycetes, plays an important role in protecting plants from phytopathogenic fungi by producing various agroactive compounds, including mycolytic enzymes particularly chitinase. The objective of this study was to investigate chitinase activity and analyze the diversity of family 19 chitinase genes of actinomycetes isolated from maize and soybean rhizosphere. Of fivety actinimycetes, Seventeen isolates exhibited chitinolytic activities and formed a hydrolytic zone around the colony with chitinolytic index ranging from 0.49±0.01 to 2.15±0.69, qualitatively tested in 0.3% chitin agar medium. Six selected isolates (ARJ 36, ARJ 81, ARJ 15, ARK 17, ARK 143, ARK 103) showed chitinase activities ranging from 0.157±0.04 to 0.440±0.09 U/mL, based on chitinolytic index. The production of chitinase was confirmed by the presence of family 19 chitinase encoding genes from Streptomyces sp. Some conserved regions and essential amino acid residues were also detected. This study suggests that chitinolytic actinomycetes isolated from maize and soybean rhizosphere can be studied further as biological control candidates for controlling phytopathogenic fungi.

Comparative molecular evolution of chitinases in ascomycota with emphasis on mycoparasitism lifestyle

Chitinases are involved in multiple aspects of fungal life cycle, such as cell wall remodelling, chitin degradation and mycoparasitism lifestyle. To improve our knowledge of the chitinase molecular evolution of Ascomycota, the gene family of 72 representatives of this phylum was identified and subjected to phylogenetic, evolution trajectory and selective pressure analyses. Phylogenetic analysis showed that the chitinase gene family size and enzyme types varied significantly, along with species evolution, especially for groups B and C. In addition, two new subgroups, C3 and C4, are recognized in group C chitinases. Random birth and death testing indicated that gene expansion and contraction occurred in most of the taxa, particularly for species in the order Hypocreales (class Sordariomycetes). From an enzyme function point of view, we speculate that group A chitinases are mainly involved in species growth and development, while the expansion of genes in group B chitinases is related to fungal mycoparasitic and entomopathogenic abilities, and, to a certain extent, the expansion of genes in group C chitinases seems to be correlated with the host range broadening of some plant-pathogenic fungi in Sordariomycetes. Further selection pressure testing revealed that chitinases and the related amino acid sites were under positive selection in the evolutionary history, especially at the nodes sharing common ancestors and the terminal branches of Hypocreales. These results give a reasonable explanation for the size and function differences of chitinase genes among ascomycetes, and provide a scientific basis for understanding the evolutionary trajectories of chitinases, particularly that towards a mycoparasitic lifestyle.

Transcriptome Analysis of Hormone-and Cuticle-Related Genes in the Development Process of Deutonymph in Tetranychus urticae

Tetranychus urticae is an important agricultural pest that feeds on more than 1100 plant species. To investigate gene expression network in development process of deutonymph, a comprehensive transcriptome analysis of different developmental time points of deutonymph in T. urticae was performed. Comparing with expression profile of 7 h, 309, 876, 2736, and 3432 differential expression genes were detected at time points 14 h, 21 h, 28 h, and 35 h, respectively. The expression dynamic analysis indicated that genes in hormone- (ecdysteroid and juvenile hormone) and cuticle- (chitin and cuticle proteins) related pathways were indispensable for development process in deutonymph. Among hormone related pathway genes, the ecdysteroid biosynthesis pathway genes were highly expressed at the growth period of development process, which is opposite to the expression patterns of juvenile hormone biosynthesis pathway genes. For cuticle related pathway genes, 13 chitinase genes were identified in the genome of T. urticae, and 8 chitinase genes were highly expressed in different time points of developmental process in the deutonymph of T. urticae. Additionally, 59 cuticle protein genes were identified from genome, and most of the cuticle protein genes were expressed in the molting period of developmental process in deutonymph. This study reveals critical genes involved in the development process of deutonymph and also provides comprehensive development transcriptome information for finding more molecular targets to control this pest.

Diversity of culturable bacteria endowed with antifungal metabolites biosynthetic characteristics associated with tea rhizosphere soil of Assam, India

Background Rhizosphere soil is a crucial niche for the diverse beneficial microbial communities in plant-microbe interactions. This study explores the antagonistic potential and diversity of the rhizosphere soil bacteria from commercial tea estates of Assam, India which comes under the Indo-Burma mega-biodiversity hotspot. Rhizosphere soil samples were collected from six different tea estates to isolate the bacteria. The bacterial isolates were subjected to evaluate for the antagonistic activity against fungal pathogens. The potential isolates were investigated for chitinase production and the presence of chitinase gene. The bacterial genetic diversity was studied by Amplified Ribosomal DNA Restriction Analysis (ARDRA) and BOX-PCR fingerprinting. Results A total of 217 rhizobacteria were isolated from tea rhizosphere soil, out of which 50 isolates exhibited the potential antagonistic activity against fungal pathogens. Among them, 12 isolates showed extracellular chitinase activity and the presence of chitinase genes. The chitinase genes were sequenced and the analysis of the sequences was performed by using PDB protein databank at the amino acid level. It showed the presence of ChiA and ChiA74 gene in the 6 most potent isolates which are involved in the hydrolysis of chitin. These isolates also exhibited antagonistic activity against all tested fungal pathogens. The diversity of 50 antagonistic bacterial isolates were analyzed through ARDRA and BOX-PCR fingerprinting. Diversity analysis and molecular identification of the rhizosphere isolates revealed that these antagonistic isolates predominantly belonged to the genus Bacillus followed by Enterobacter, Serratia, Lysinibacillus, Pseudomonas, and Burkholderia. Conclusion The present study establishes that rhizobacteria isolated from the poorly explored tea rhizosphere soil could be a rich reservoir for the investigation of potential antagonistic bacterial candidates for sustainable agricultural and industrial applications.