Chitin Catabolism Research Articles

Genes of Streptomyces globisporus 1912-4Crt Encoding Chitin Catabolism Enzymes

Polysaccharide chitin is one of the most common biopolymers in nature. Chitin (when used as the sole source of energy, carbon, and nitrogen) has been shown to be a substrate sufficient to enable the growth and synthesis of secondary metabolites by the S. griseus NCIMB 8136 strain and some other streptomycetes. The species Streptomyces globisporus and S. griseus belong to the same lower hierarchical taxon (S. griseus clade). The aim was to find in the S. globisporus 1912-4Crt genome genes encoding proteins that are necessary for chitin fermentation and transmembrane transport of resulting products. Methods. The object of the study was a sequence of the S. globisporus 1912-4Crt genome (reference NZ_QWFA01000000.1, GenBank) on the server of NCBI (The National Center for Biotechnology Information). Streptomycete S. globisporus 1912 and its variants are producers of antibiotic landomycin E and carotenoids. Search for and analysis of nucleotide and amino acid sequences were performed using programs BLAST (Basic Local Alignment Search Tool) on the aforementioned server. Results. A set of genes that determine enzymes of chitin catabolism and Ngc-transporter proteins were identified in the S. globisporus 1912-4Crt genome. Genes encoding 10 endo-acting chitinases (from the GH18 and GH19 families) and 2 exo-acting hydrolases (GH20) were identified in the S. globisporus 1912-4Crt sequence. Several genes determining deacetylases that deacetylate both chitin and oligosaccharides were found in the genome of the strain. One gene that determines endo-acting chitosanase from the GH75 family was discovered there. The ability of the wild-type strain S. globisporus 1912 and a number of its variants (including S. globisporus 1912-4Crt) to ferment chitin was proven in vitro. Conclusions. A set of genes sufficient for chitin assimilation was established in the S. globisporus 1912-4Crt genome sequence. Streptomycetes from different clades (for example, strains of S. coelicolor (S. albidoflavus group) and S. griseus, S. globisporus (S. griseus group)) containing different complexes of chitinolytic enzymes could be suggested.

A novel endo-type chitinase possessing chitobiase activity derived from the chitinolytic bacterium, Chitiniphilus shinanonensis SAY3T.

One of the chitinases (ChiG) derived from the chitinolytic bacterium Chitiniphilus shinanonensis SAY3T exhibited chitobiase activity cleaving dimers of N-acetyl-D-glucosamine (GlcNAc) into monomers, which is not detected in typical endo-type chitinases. Analysis of the reaction products for GlcNAc hexamers revealed that all the five internal glycosidic bonds were cleaved at the initial stage. The overall reaction catalyzed by chitobiases toward GlcNAc dimers was similar to that catalyzed by N-acetyl-D-glucosaminidases (NAGs). SAY3 possesses two NAGs (ChiI and ChiT) that are thought to be important in chitin catabolism. Unexpectedly, a triple gene-disrupted mutant (ΔchiIΔchiTΔchiG) was still able to grow on synthetic medium containing GlcNAc dimers or powdered chitin, similar to the wild-type SAY3, although it exhibited only 3% of total cellular NAG activity compared to the wild-type. This indicates the presence of unidentified enzyme(s) capable of supporting normal bacterial growth on the chitin medium by NAG activity compensation.

Enzymatically hydrolyzed fluorescence-based chemical probe enables in situ mapping of chitinase activity in the rhizosphere

Chitin is an insoluble and ubiquitous soil biopolymer, estimated to be the second most abundant organic soil biopolymer on Earth. Despite its abundance, role as a source of C and N in soil, and importance to ecosystem function, further research is required to elucidate key controls on chitin breakdown under varying environmental conditions. Previous work highlights the important role rhizosphere microbiomes and root exudates can play in chitin catabolism. To enable mapping of chitinase activity within the highly heterogeneous and spatially organized rhizosphere, we designed and synthesized an enzymatically activated fluorogenic substrate, chitotriose-TokyoGreen (chitotriose-TG), by incorporating a fluorescein derivative (TG) onto the trimeric unit of chitin. This non-fluorescent substrate is selectively hydrolyzed by chitinase to release TG and yield a fluorescence signal, which can be used to spatially image and measure chitinase activity in the rhizosphere. To demonstrate the application of this technique, we grew switchgrass (Panicum virgatum) in rhizoboxes amended with a horizontal layer supplemented with chitin. We extracted mobile proteins from the rhizobox using a nitrocellulose membrane blotting technique which offers non-destructive enzyme extraction while preserving the 2D spatial position of the enzymes. We then subjected these membranes to the synthesized chitotriose-TG stain to spatially visualize the distribution of chitinase activity within the rhizosphere. We observed increased chitinase activity near switchgrass roots and higher activity within the soil zone enriched in chitin, showing an adaptive response of chitinase production with spatial focusing in areas of higher chitin abundance. Thus, the enzyme extraction and visualization strategy we describe here can enhance efforts to better understand spatial controls on chitin breakdown in rhizosphere, further elucidating the role of chitin as a C and N source in these systems.

Functional importance of groups I and II chitinases in cuticle chitin turnover during molting in a wood-boring beetle, Monochamus alternatus

Insects must periodically replace their old cuticle/exoskeleton with a new one in a process called molting or ecdysis to allow for continuous growth through sequential developmental stages. Many RNA interference (RNAi) studies have demonstrated that certain chitinases (CHTs) play roles in this vital physiological event because knockdown of these CHT genes resulted in developmental arrest during the ensuing molting period in several insect species. In this research we analyzed the functions of group I (MaCHT5) and group II (MaCHT10) CHT genes in molting of the Japanese pine sawyer, Monochamus alternatus, an important forest pest known as a major vector of the pinewood nematode. Real-time qPCR revealed that these two CHT genes differ in their expression patterns during late stages of development. Depletion of either MaCHT5 or MaCHT10 transcripts by RNAi resulted in lethal larval-pupal and pupal-adult molting defects depending on the double-stranded RNA (dsRNA) injection timing during development. The insects were unable to shed their old cuticle and died. Furthermore, transmission electron microscopic analysis revealed that, unlike dsEGFP-treated controls, dsMaCHT5- and dsMaCHT10-treated pharate adults exhibited a failure of degradation of the endocuticular layer of their old pupal cuticle, retaining nearly intact horizontal chitinous laminae and vertical pore canal fibers. Both enzymes were indispensable for complete turnover of the chitinous old endocuticle, which is critical for insect molting. The possible functions of two spliced variants of MaCHT10, namely, MaCHT10a and MaCHT10b, are also discussed. Our results add to the knowledge base for further functional studies of insect chitin catabolism by revealing the relative importance of both MaCHT5 and MaCHT10 in chitin turnover with subtle differences in their action. These essential genes and their encoded proteins are potential targets to manipulate for controlling populations of M. alternatus and other pest insects.

Genomic analysis of Marinimicrobium sp. C6131 reveals its genetic potential involved in chitin metabolism.

Marinimicrobium sp. C6131, which had the ability to degrade chitin, was isolated from deep-sea sediment of the southwest Indian Ocean. Here, the genome of strain C6131 was sequenced and the chitin metabolic pathways were constructed. The genome contained a circular chromosome of 4,207,651bp with a G+C content of 58.50%. A total of 3471 protein-coding sequences were predicted. Gene annotation and metabolic pathway reconstruction showed that strain C6131 possessed genes and two metabolic pathways involved in chitin catabolism: the hydrolytic chitin utilization pathway initiated by chitinases and the oxidative chitin utilization pathway initiated by lytic polysaccharide monooxygenases. Chitin is the most abundant polysaccharide in the ocean. Degradation and recycling of chitin driven by marine bacteria are crucial for biogeochemical cycles of carbon and nitrogen in the ocean. The genomic information of strain C6131 revealed its genetic potential involved in chitin metabolism. The strain C6131 could grow with colloidal chitin as the sole carbon source, indicating that these genes would have functions in chitin degradation and utilization. The genomic sequence of Marinimicrobium sp. C6131 could provide fundamental information for future studies on chitin degradation, and help to improve our understanding of the chitin degradation process in deep-sea environments.

Physiological and Transcriptomic Responses of Illicium difengpi to Drought Stress

Illicium difengpi Kib and Kim, an endangered plant unique to karst areas in China, has evolved an extremely high tolerance to arid environments. To elucidate the molecular mechanisms of the response to drought stress in I. difengpi, physiological index determination and transcriptome sequencing experiments were conducted in biennial seedlings grown under different soil moisture conditions (70~80%, 40~50% and 10~20%). With increasing drought stress, the leaf chlorophyll content decreased, while the proline (Pro), soluble sugar (SS) and malondialdehyde (MDA) contents increased; superoxide dismutase (SOD) and peroxidase (POD) activities also increased. Transcriptome sequencing and pairwise comparisons of the treatments revealed 2489, 4451 and 753 differentially expressed genes (DEGs) in CK70~80 vs. XP40~50, CK70~80 vs. XP10~20 and XP40~50 vs. XP10~20, respectively. These DEGs were divided into seven clusters according to their expression trends, and the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment results of different clusters indicated that genes in the hormone signal transduction and osmotic regulation pathways were greatly activated under mild drought stress. When drought stress increased, the DEGs related to membrane system and protein modification and folding were all upregulated; simultaneously, chitin catabolism- and glycolysis/gluconeogenesis-related genes were continuously upregulated throughout drought stress, while the genes involved in photosynthesis were downregulated. Here, 244 transcription factors derived from 10 families were also identified. These results lay a foundation for further research on the adaptation of I. difengpi to arid environments in karst areas and the establishment of a core regulatory relationship in its drought resistance mechanism.

Genetic drift and host-adaptive features likely underlie cladogenesis of insect-associated Lachnospiraceae.

Phylogenetic and functional group analyses of the genomes of anaerobic bacteria isolated from Periplaneta americana digestive tracts suggest that they represent novel Lachnospiraceae genera. PAL113 and PAL227 isolate genomes encoded short-chain fatty acid biosynthetic pathways and plant fiber and chitin catabolism and other carbohydrate utilization genes common in related Lachnospiraceae species, yet the presence of operons containing flagellar assembly pathways was among several distinguishing features. In general, PAL113 and PAL227 isolates encode an array of gene products that will enable them to thrive in the insect gut environment and potentially play a role in host diet processing. We hypothesize that the cladogenesis of these isolates can be a result of their oxygen sensitivity and reliance upon the host for dispersal and genetic drift and not necessarily a result of an ongoing mutualism.

The Role of Chitooligosaccharidolytic β-N-Acetylglucosamindase in the Molting and Wing Development of the Silkworm Bombyx mori.

The insect glycoside hydrolase family 20 β-N-acetylhexosaminidases (HEXs) are key enzymes involved in chitin degradation. In this study, nine HEX genes in Bombyx mori were identified by genome-wide analysis. Bioinformatic analysis based on the transcriptome database indicated that each gene had a distinct expression pattern. qRT-PCR was performed to detect the expression pattern of the chitooligosaccharidolytic β-N-acetylglucosaminidase (BmChiNAG). BmChiNAG was highly expressed in chitin-rich tissues, such as the epidermis. In the wing disc and epidermis, BmChiNAG has the highest expression level during the wandering stage. CRISPR/Cas9-mediated BmChiNAG deletion was used to study the function. In the BmChiNAG-knockout line, 39.2% of female heterozygotes had small and curly wings. The ultrastructure of a cross-section showed that the lack of BmChiNAG affected the stratification of the wing membrane and the formation of the correct wing vein structure. The molting process of the homozygotes was severely hindered during the larva to pupa transition. Epidermal sections showed that the endocuticle of the pupa was not degraded in the mutant. These results indicate that BmChiNAG is involved in chitin catabolism and plays an important role in the molting and wing development of the silkworm, which highlights the potential of BmChiNAG as a pest control target.

Discovery of Biphenyl-Sulfonamides as Novel β-N-Acetyl-d-Hexosaminidase Inhibitors via Structure-Based Virtual Screening.

Novel insecticidal targets are always in demand due to the development of resistance. OfHex1, a β-N-acetyl-d-hexosaminidase identified in Ostrinia furnacalis (Asian corn borer), is involved in insect chitin catabolism and has proven an ideal target for insecticide development. In this study, structure-based virtual screening, structure simplification, and biological evaluation are used to show that compounds with a biphenyl-sulfonamide skeleton have great potential as OfHex1 inhibitors. Specifically, compounds 10k, 10u, and 10v have Ki values of 4.30, 3.72, and 4.56 μM, respectively, and thus, they are more potent than some reported nonglycosyl-based inhibitors such as phlegmacin B1 (Ki = 26 μM), berberine (Ki = 12 μM), 2 (Ki = 11.2 μM), and 3 (Ki = 28.9 μM). Furthermore, inhibitory kinetic assessments reveal that the target compounds are competitive inhibitors with respect substrate, and based on toxicity predictions, most of them have potent drug properties. The obtained results indicate that the biphenyl-sulfonamide skeleton characterized by simple chemical structure, synthetic tractability, potent activity, and low toxicity has potential for further development in pest management targeting OfHex1.

Functional metagenomics reveals differential chitin degradation and utilization features across free-living and host-associated marine microbiomes

BackgroundChitin ranks as the most abundant polysaccharide in the oceans yet knowledge of shifts in structure and diversity of chitin-degrading communities across marine niches is scarce. Here, we integrate cultivation-dependent and -independent approaches to shed light on the chitin processing potential within the microbiomes of marine sponges, octocorals, sediments, and seawater.ResultsWe found that cultivatable host-associated bacteria in the genera Aquimarina, Enterovibrio, Microbulbifer, Pseudoalteromonas, Shewanella, and Vibrio were able to degrade colloidal chitin in vitro. Congruent with enzymatic activity bioassays, genome-wide inspection of cultivated symbionts revealed that Vibrio and Aquimarina species, particularly, possess several endo- and exo-chitinase-encoding genes underlying their ability to cleave the large chitin polymer into oligomers and dimers. Conversely, Alphaproteobacteria species were found to specialize in the utilization of the chitin monomer N-acetylglucosamine more often. Phylogenetic assessments uncovered a high degree of within-genome diversification of multiple, full-length endo-chitinase genes for Aquimarina and Vibrio strains, suggestive of a versatile chitin catabolism aptitude. We then analyzed the abundance distributions of chitin metabolism-related genes across 30 Illumina-sequenced microbial metagenomes and found that the endosymbiotic consortium of Spongia officinalis is enriched in polysaccharide deacetylases, suggesting the ability of the marine sponge microbiome to convert chitin into its deacetylated—and biotechnologically versatile—form chitosan. Instead, the abundance of endo-chitinase and chitin-binding protein-encoding genes in healthy octocorals leveled up with those from the surrounding environment but was found to be depleted in necrotic octocoral tissue. Using cultivation-independent, taxonomic assignments of endo-chitinase encoding genes, we unveiled previously unsuspected richness and divergent structures of chitinolytic communities across host-associated and free-living biotopes, revealing putative roles for uncultivated Gammaproteobacteria and Chloroflexi symbionts in chitin processing within sessile marine invertebrates.ConclusionsOur findings suggest that differential chitin degradation pathways, utilization, and turnover dictate the processing of chitin across marine micro-niches and support the hypothesis that inter-species cross-feeding could facilitate the co-existence of chitin utilizers within marine invertebrate microbiomes. We further identified chitin metabolism functions which may serve as indicators of microbiome integrity/dysbiosis in corals and reveal putative novel chitinolytic enzymes in the genus Aquimarina that may find applications in the blue biotechnology sector.4kjGuZRPCftKy_HwBHqQX8Video abstract

Activity-Based Protein Profiling of Chitin Catabolism.

The microbial catabolism of chitin, an abundant and ubiquitous environmental organic polymer, is a fundamental cog in terrestrial and aquatic carbon and nitrogen cycles. Despite the importance of this critical bio-geochemical function, there is a limited understanding of the synergy between the various hydrolytic and accessory enzymes involved in chitin catabolism. To address this deficit, we synthesized activity-based probes (ABPs) designed to target active chitinolytic enzymes by modifying the chitin subunits N-acetyl glucosamine and chitotriose. The ABPs were used to determine the active complement of chitinolytic enzymes produced over time by the soil bacterium Cellvibrio japonicus treated with various C substrates. We demonstrate the utility of these ABPs in determining the synergy between various enzymes involved in chitin catabolism. The strategy can be used to gain molecular-level insights that can be used to better understand microbial roles in soil bio-geochemical cycling in the face of a changing climate.

Compendium of Colletotrichum graminicola responsive infection-induced transcriptomic shifts in the maize

Pathogen-induced biotic stress stands as the foremost factor undermining global agricultural productivity, and comparative transcriptomics studies are ideal for zeroing in on processes remodeled in response to the pathogen. We examined the maize-Colletotrichum graminicola pathosystem to discern the host-deployed defensive program towards this fungal pathogen. Of a total of 36,058 transcripts identified, 306 show significant differential expression (5% FDR). The differentially expressed genes are implicated in pathogenesis, stress signaling, disease defense, cell wall reinforcement, signal perception, and transduction. Comparative Gene ontological enrichment analysis of the differentially expressed genes revealed enrichment in terms involved in fungal defense, such as aminoglycan metabolism and catabolism, chitin metabolism and catabolism, cell wall macromolecule metabolic and catabolic process, response to stress and hydrolyzing o-glycosyl compounds. Subsequent analysis highlighted transcript mapping to many prominent defensive pathways like phenylpropanoid, plant-pathogen interaction, ubiquitin proteolysis, and biosynthesis of secondary metabolites. We observed over-expression of the lysM domain containing possible novel chitin elicitor binding protein (CEBiP) homolog C0PL67 (Uniprot identifier), and GRMZM2G056329, a Casparian strip membrane domain-like protein (CASP-like), previously uncharacterized in the maize-Colletotrichum interface, that could potentially act as a target gene for subduing pathogenesis. Further, sequence analysis of the differentially expressed genes resulted in the identification of possible, pathogenesis-relevant, de-novo motifs and effectors.

Repetitive somatic embryogenesis induced cytological and proteomic changes in embryogenic lines of Pseudotsuga menziesii [Mirb.

BackgroundTo explore poorly understood differences between primary and subsequent somatic embryogenic lines of plants, we induced secondary (2ry) and tertiary (3ry) lines from cotyledonary somatic embryos (SEs) of two Douglas-fir genotypes: SD4 and TD17. The 2ry lines exhibited significantly higher embryogenic potential (SE yields) than the 1ry lines initiated from zygotic embryos (SD4, 2155 vs 477; TD17, 240 vs 29 g− 1 f.w.). Moreover, we observed similar differences in yield between 2ry and 3ry lines of SD4 (2400 vs 3921 g− 1 f.w.). To elucidate reasons for differences in embryogenic potential induced by repetitive somatic embryogenesis we then compared 2ry vs 1ry and 2ry vs 3ry lines at histo-cytological (using LC-MS/MS) and proteomic levels.ResultsRepetitive somatic embryogenesis dramatically improved the proliferating lines’ cellular organization (genotype SD4’s most strongly). Frequencies of singulated, bipolar SEs and compact polyembryogenic centers with elongated suspensors and apparently cleavable embryonal heads increased in 2ry and (even more) 3ry lines. Among 2300–2500 identified proteins, 162 and 228 were classified significantly differentially expressed between 2ry vs 1ry and 3ry vs 2ry lines, respectively, with special emphasis on “Proteolysis” and “Catabolic process” Gene Ontology categories. Strikingly, most of the significant proteins (> 70%) were down-regulated in 2ry relative to 1ry lines, but up-regulated in 3ry relative to 2ry lines, revealing a down-up pattern of expression. GO category enrichment analyses highlighted the opposite adjustments of global protein patterns, particularly for processes involved in chitin catabolism, lignin and L-phenylalanine metabolism, phenylpropanoid biosynthesis, oxidation-reduction, and response to karrikin. Sub-Network Enrichment Analyses highlighted interactions between significant proteins and both plant growth regulators and secondary metabolites after first (especially jasmonic acid, flavonoids) and second (especially salicylic acid, abscisic acid, lignin) embryogenesis cycles. Protein networks established after each induction affected the same “Plant development” and “Defense response” biological processes, but most strongly after the third cycle, which could explain the top embryogenic performance of 3ry lines.ConclusionsThis first report of cellular and molecular changes after repetitive somatic embryogenesis in conifers shows that each cycle enhanced the structure and singularization of EMs through modulation of growth regulator pathways, thereby improving the lines’ embryogenic status.

Chitins and chitinase activity in airway diseases

Chitin, one of the most abundant biopolymers on Earth, is bound and degraded by chitinases, specialized enzymes that are similarly widespread in nature. Chitin catabolism affects global carbon and nitrogen cycles through a host of diverse biological processes, but recent work has focused attention on systems of chitin recognition and degradation conserved in mammals, connecting an ancient pathway of polysaccharide processing to human diseases influenced by persistent immune triggering. Here we review current advances in our understanding of how chitin-chitinase interactions affect mucosal immune feedback mechanisms essential to maintaining homeostasis and organ health.

Sex-specific transcript expression in the hepatopancreas of the banana shrimp (Fenneropenaeus merguiensis)

The banana shrimp, Fenneropenaeus merguiensis, is a commercially important species of marine crustacean that, like other penaeids, exhibit a female-superior sexual dimorphism with respect to growth rate, making this species an attractive candidate for all-female (monosex) culture. To gain insight into the molecular mechanisms underpinning this characteristic, an analysis of gene expression was performed to compare digestive gland tissue from 14 male and 14 female banana shrimp (n = 28) larger than 10 g body weight. Female banana shrimp samples were found to have an increased expression of transcripts coding for chitin catabolism and binding. A novel transcript was also identified that displayed a distinct female-specific expression. Weighted gene co-expression network analysis (WGCNA) from female samples revealed that this novel transcript was highly connected within a gene module containing an abundance of transcripts with homology to transcription factors and genes associated with growth regulation.

Glucose-Specific Enzyme IIA of the Phosphoenolpyruvate:Carbohydrate Phosphotransferase System Modulates Chitin Signaling Pathways in Vibrio cholerae.

In Vibrio cholerae, the genes required for chitin utilization and natural competence are governed by the chitin-responsive two-component system (TCS) sensor kinase ChiS. In the classical TCS paradigm, a sensor kinase specifically phosphorylates a cognate response regulator to activate gene expression. However, our previous genetic study suggested that ChiS stimulates the non-TCS transcriptional regulator TfoS by using mechanisms distinct from classical phosphorylation reactions (S. Yamamoto, J. Mitobe, T. Ishikawa, S. N. Wai, M. Ohnishi, H. Watanabe, and H. Izumiya, Mol Microbiol 91:326-347, 2014, https://doi.org/10.1111/mmi.12462). TfoS specifically activates the transcription of tfoR, encoding a small regulatory RNA essential for competence gene expression. Whether ChiS and TfoS interact directly remains unknown. To determine if other factors mediate the communication between ChiS and TfoS, we isolated transposon mutants that turned off tfoR::lacZ expression but possessed intact chiS and tfoS genes. We demonstrated an unexpected association of chitin-induced signaling pathways with the glucose-specific enzyme IIA (EIIAglc) of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) for carbohydrate uptake and catabolite control of gene expression. Genetic and physiological analyses revealed that dephosphorylated EIIAglc inactivated natural competence and tfoR transcription. Chitin-induced expression of the chb operon, which is required for chitin transport and catabolism, was also repressed by dephosphorylated EIIAglc Furthermore, the regulation of tfoR and chb expression by EIIAglc was dependent on ChiS and intracellular levels of ChiS were not affected by disruption of the gene encoding EIIAglc These results define a previously unknown connection between the PTS and chitin signaling pathways in V. cholerae and suggest a strategy whereby this bacterium can physiologically adapt to the existing nutrient status.IMPORTANCE The EIIAglc protein of the PTS coordinates a wide variety of physiological functions with carbon availability. In this report, we describe an unexpected association of chitin-activated signaling pathways in V. cholerae with EIIAglc The signaling pathways are governed by the chitin-responsive TCS sensor kinase ChiS and lead to the induction of chitin utilization and natural competence. We show that dephosphorylated EIIAglc inhibits both signaling pathways in a ChiS-dependent manner. This inhibition is different from classical catabolite repression that is caused by lowered levels of cyclic AMP. This work represents a newly identified connection between the PTS and chitin signaling pathways in V. cholerae and suggests a strategy whereby this bacterium can physiologically adapt to the existing nutrient status.

Characterization of 47 Cys2 -His2 zinc finger proteins required for the development and pathogenicity of the rice blast fungus Magnaporthe oryzae.

The Cys2 -His2 (C2H2) zinc finger protein family is the second-largest family of transcription factors (TFs) in Magnaporthe oryzae, the causal fungus responsible for the destructive rice blast disease. However, little is known about the roles of most C2H2 TFs in the development and pathogenicity of M.oryzae. The roles of 47 C2H2 genes in development and pathogenicity were investigated by gene deletion in M.oryzae. The TF-dependent genes in mycelia or appressoria were analyzed with RNA sequencing and quantitative PCR (qPCR). Forty-four C2H2 genes are involved in growth (20 genes), conidiation (28 genes), appressorium formation (four genes) and pathogenicity (22 genes) in M.oryzae. Of these, MGG_14931, named as VRF1, is required for pathogenicity, specifically controlling appressorium maturation by affecting the expression of genes related to appressorial structure and function, including melanin biosynthesis, chitin catabolism, lipid metabolism, proteolysis, transmembrane transport, and response to oxidative stress; MGG_01776, named as VRF2, is required for plant penetration and invasive growth; conidiation-related gene CON7 is required for conidial differentiation; and MoCREA, encoding a carbon catabolite repression protein, is a novel repressor of lipid catabolism when glucose obtainable in M.oryzae. This study provides many insights into the regulation of growth, asexual development, appressorium formation, and pathogenicity by C2H2 TFs in M.oryzae.

Cloning and functional expression of a chitinase cDNA from the apple leaf miner moth Lithocolletis ringoniella.

Insect chitinase plays essential roles in chitin catabolism involved in digestion and molting during insect development. In the current work, we cloned a chitinase cDNA, LrCht5, from the apple leaf miner moth Lithocolletis ringoniella and characterized its amino acid sequence and protein properties. The L. ringoniella chitinase cDNA was 2136 bp in length with an open reading frame of 1737 bp that encodes a polypeptide of 579 amino acid residues with a predicted molecular mass of 64.4 kDa and pI of 5.49. The catalytic domain has several phosphorylation and glycosylation sites. The recombinant LrCht5 was expressed in Escherichia coli and the Spodoptera frugiperda cell line Sf9, and the LrCht5 expressed in insect cells exhibited chitinolytic activity. LrCht5 was most stable at pH 6.0 and 45°C. This work has potential application in the development of novel and more specific synthetic chitinase inhibitors for use as bioinsecticides.

Proteomic analysis in caged Mediterranean crab (Carcinus maenas) and chemical contaminant exposure in Téboulba Harbour, Tunisia

This study uses proteomics approach to assess the toxic effects of contaminants in the Mediterranean crab (Carcinus maenas) after transplantation into Téboulba fishing harbour. High levels of aliphatic and aromatic hydrocarbons were detected in sediments. Although their effects on vertebrates are well described, little is known about their early biological effects in marine invertebrates under realistic conditions. Protein expression profiles of crabs caged for 15, 30 and 60 days were compared to unexposed animals. Nineteen proteins with significant expression differences were identified by capLC-µESI-IT MS/MS and homology search on databases. Differentially expressed proteins were assigned to five different categories of biological function including: (1) chitin catabolism, (2) proteolysis, (3) exoskeleton biosynthesis, (4) protein folding and stress response, and (5) transport. The proteins showing major expression changes in C. maenas after different caging times may be considered as novel molecular biomarkers for effectively biomonitoring aquatic environment contamination.

Comparative Proteomics of Primary and Secondary Lutoids Reveals that Chitinase and Glucanase Play a Crucial Combined Role in Rubber Particle Aggregation in Hevea brasiliensis

Lutoids are specific vacuole-based organelles within the latex-producing laticifers in rubber tree Hevea brasiliensis. Primary and secondary lutoids are found in the primary and secondary laticifers, respectively. Although both lutoid types perform similar roles in rubber particle aggregation (RPA) and latex coagulation, they vary greatly at the morphological and proteomic levels. To compare the differential proteins and determine the shared proteins of the two lutoid types, a proteomic analysis of lutoid membranes and inclusions was performed, revealing 169 proteins that were functionally classified into 14 families. Biological function analysis revealed that most of the proteins are involved in pathogen defense, chitin catabolism, and proton transport. Comparison of the gene and protein changed patterns and determination of the specific roles of several main lutoid proteins, such as glucanase, hevamine, and hevein, demonstrated that Chitinase and glucanase appeared to play crucial synergistic roles in RPA. Integrative analysis revealed a protein-based metabolic network mediating pH and ion homeostasis, defense response, and RPA in lutoids. From these findings, we developed a modified regulation model for lutoid-mediated RPA that will deepen our understanding of potential mechanisms involved in lutoid-mediated RPA and consequent latex coagulation.