Chitin Synthase Genes Research Articles

Identification and detection of a novel point mutation in the Chitin Synthase gene of Culex pipiens associated with diflubenzuron resistance.

BackgroundDiflubenzuron (DFB) is one of the most used insecticides in mosquito larval control including that of Culex pipiens, the proven vector of the recent West Nile Virus epidemics in Europe. Two mutations (I1043L and I1043M) in the chitin synthase (CHS) putative binding site of DFB have been previously reported in Cx. pipiens from Italy and associated with high levels of resistance against this larvicide.Methodology/Principal findingsHere we report the identification of a third mutation at the same I1043 position of the CHS gene resulting in the substitution of Isoleucine to Phenylalanine (I1043F). This mutation has also been found in agricultural pests and has been functionally validated with genome editing in Drosophila, showing to confer striking levels (>15,000 fold) of DFB resistance. The frequency of the I1043F mutation was found to be substantially higher in Cx. pipiens mosquitoes surviving DFB doses largely exceeding the recommended field dose, raising concerns about the future efficient use of this insecticide. We monitored the presence and frequency of DFB mutations in Cx. pipiens mosquitoes from several Mediterranean countries, including Italy, France, Greece, Portugal and Israel. Among the Cx. pipiens populations collected in Northern Italy all but one had at least one of the three DFB mutations at allele frequencies reaching 93.3% for the I1043M, 64.8% for the I1043L and 10% for the I1043F. The newly reported I1043F mutation was also identified in two heterozygote individuals from France (4.2% allelic frequency). In contrast to Italy and France, no DFB resistant mutations were identified in the Cx. pipiens mosquitoes sampled from Greece, Portugal and Israel.Conclusions/SignificanceThe findings of our study are of major concern for mosquito control programs in Europe, that rely on the use of a limited number of available larvicides, and highlight the necessity for the development of appropriate Insecticide Resistance Management (IRM) programs, to ensure the sustainable use of DFB.

Identification and Functional Analysis of Two Chitin Synthase Genes in the Common Cutworm, Spodoptera litura.

Chitin is one the main components of the insect cuticle, and chitin synthase (CHS) is an important enzyme required for chitin formation. CHS has been characterized in various insect species, but the structure and biochemical properties in Spodoptera litura have not been determined. In this study, we identified two CHS genes, SlCHS1 and SlCHS2, which encode proteins with 1565 and 1520 amino acid residues, respectively. Transcriptional analysis suggested that SlCHS1 has a high expression level in the integument whereas SlCHS2 showed the highest expression level in the midgut. During S. litura growth and development, SlCHS1 and SlCHS2 were both predominantly expressed in the fourth-instar larval stage. In addition, the expression of SlCHS1 and SlCHS2 could be induced by 20-hydroxyecdysone (20E). Silencing of SlCHS1 by RNA interference significantly inhibited the pupation and molting of S. litura larvae (RNAi), while knockdown of SlCHS2 had no significant effects on the S. litura phenotype. These results may provide a new molecular target for control of S. litura.

Effects of 5'-3' Exonuclease Xrn1 on Cell Size, Proliferation and Division, and mRNA Levels of Periodic Genes in Cryptococcus neoformans.

Cell size affects almost all biosynthetic processes by controlling the size of organelles and disrupting the nutrient uptake process. Yeast cells must reach a critical size to be able to enter a new cell cycle stage. Abnormal changes in cell size are often observed under pathological conditions such as cancer disease. Thus, cell size must be strictly controlled during cell cycle progression. Here, we reported that the highly conserved 5′-3′ exonuclease Xrn1 could regulate the gene expression involved in the cell cycle pathway of Cryptococcus neoformans. Chromosomal deletion of XRN1 caused an increase in cell size, defects in cell growth and altered DNA content at 37 °C. RNA-sequencing results showed that the difference was significantly enriched in genes involved in membrane components, DNA metabolism, integration and recombination, DNA polymerase activity, meiotic cell cycle, nuclear division, organelle fission, microtubule-based process and reproduction. In addition, the proportion of the differentially expressed periodic genes was up to 19.8% when XRN1 was deleted, including cell cycle-related genes, chitin synthase genes and transcription factors, indicating the important role of Xrn1 in the control of cell cycle. This work provides insights into the roles of RNA decay factor Xrn1 in maintaining appropriate cell size, DNA content and cell cycle progression.

Breakpoint: Cell Wall and Glycoproteins and their Crucial Role in the Phytopathogenic Fungi Infection.

The cell wall that surrounds fungal cells is essential for their survival, provides protection against physical and chemical stresses, and plays relevant roles during infection. In general, the fungal cell wall is composed of an outer layer of glycoprotein and an inner skeletal layer of β-glucans or α- glucans and chitin. Chitin synthase genes have been shown to be important for septum formation, cell division and virulence. In the same way, chitin can act as a potent elicitor to activate defense response in several plant species; however, the fungi can convert chitin to chitosan during plant infection to evade plant defense mechanisms. Moreover, α-1,3-Glucan, a non-degradable polysaccharide in plants, represents a key feature in fungal cell walls formed in plants and plays a protective role for this fungus against plant lytic enzymes. A similar case is with β-1,3- and β-1,6-glucan which are essential for infection, structure rigidity and pathogenicity during fungal infection. Cell wall glycoproteins are also vital to fungi. They have been associated with conidial separation, the increase of chitin in conidial cell walls, germination, appressorium formation, as well as osmotic and cell wall stress and virulence; however, the specific roles of glycoproteins in filamentous fungi remain unknown. Fungi that can respond to environmental stimuli distinguish these signals and relay them through intracellular signaling pathways to change the cell wall composition. They play a crucial role in appressorium formation and penetration, and release cell wall degrading enzymes, which determine the outcome of the interaction with the host. In this review, we highlight the interaction of phypatophogen cell wall and signaling pathways with its host and their contribution to fungal pathogenesis.

Moniliophthora perniciosa development: key genes involved in stress-mediated cell wall organization and autophagy

Moniliophthora perniciosa is a basidiomycete responsible for the witches' broom disease in cacao (Theobroma cacao L.). Chitin synthase (CHS), chitinase (CHIT) and autophagy (ATG) genes have been associated to stress response preceding the formation of basidiocarp. An analysis of literature mining, interactomics and gene expression was developed to identify the main proteins related to development, cell wall organization and autophagy in M. perniciosa. TORC2 complex elements were identified and were involved in the response to the nutrient starvation during the fungus development stages preceding the basidiocarp formation. This complex interacted with target proteins related to cell wall synthesis and to polarization and cell division (FKS1, CHS, CDC42, ROM2). Autolysis and autophagy processes were associated to CHIT2, ATG8 and to the TORC1 complex (TOR1 and KOG1), which is central in the upstream signalization of the stress response due to nutrient starvation and growth regulation. Other important elements that participate to steps preceding basidiocarp formation were also identified (KOG1, SSZ1, GDI1, FKS1, CCD10, CKS1, CDC42, RHO1, AVO1, BAG7). Similar gene expression patterns during fungus reproductive structure formation and when treated by rapamycin (a nutritional related-autophagy stress agent) were observed: cell division related-genes were repressed while those related to autolysis/autophagy were overexpressed.

Neev, a novel long non-coding RNA, is expressed in chaetoblasts during regeneration of Eisenia fetida

Eisenia fetida, the common vermicomposting earthworm, shows robust regeneration of posterior segments removed by amputation. During the period of regeneration, the newly formed tissue initially contains only undifferentiated cells but subsequently differentiates into a variety of cell types including muscle, nerve and vasculature. Transcriptomics analysis, reported previously, provided a number of candidate non-coding RNAs that were induced during regeneration. We found that one such long non-coding RNA (lncRNA) is expressed in the skin, only at the base of newly formed chaetae. The spatial organization and precise arrangement of the regenerating chaetae and the cells expressing the lncRNA on the ventral side clearly support a model wherein the regenerating tissue contains a zone of growth and cell division at the tip and a zone of differentiation at the site of amputation. The temporal expression pattern of the lncRNA, named Neev, closely resembled the pattern of chitin synthase genes, implicated in chaetae formation. We found that the lncRNA has 49 sites for binding a set of four microRNAs (miRNAs) while the chitin synthase 8 mRNA has 478 sites. The over-representation of shared miRNA sites suggests that lncRNA Neev may act as a miRNA sponge to transiently de-repress chitin synthase 8 during formation of new chaetae in the regenerating segments of Eisenia fetida.

Analysis of the Gene Expression and RNAi-Mediated Knockdown of Chitin Synthase from Leaf-Cutting Ant Atta sexdens

Chitin synthase (CHS) is the enzyme specifically associated with chitin synthesis, an important component of diverse organisms including insects. Two alternative spliced transcripts of the CHS gene (AsCHS-A1 and AsCHS-A2) were identified by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) during the development of the leaf-cutting ant Atta sexdens. Expression profiles of AsCHS-A transcripts increased from larva to pupae and decay in workers. Phylogenetic analysis showed both transcripts are classified within class A insect CHSs. AsCHS-A1 showed the highest expression level in larvae and pupae, while AsCHS-A2 is the main CHS transcript in workers. Our results suggest that these variants should be under regulation of different promoters. AsCHS-A1 has topology expected for insect CHSs, while the predicted AsCHS-A2 topology, with a missing A domain, is similar to some fungal CHSs. CHS-B (class B) was not identified in A. sexdens transcriptome. Ribonucleic acid interference (RNAi)-mediated gene silencing in pupae revealed that low reduction in CHS transcript levels (18%) was enough to cause morphological changes in the pupa exoskeleton impairing the process of cuticle sclerotization. To our knowledge, this work was the first to use and to show the feasibility of using RNA interference techniques on leaf-cutting ants.

Identification and Characterization of the Chitin Synthase Genes From the Fish Pathogen Saprolegnia parasitica.

Saprolegnia parasitica is a pathogenic oomycete responsible for severe fish infections. Despite its low abundance in the cell wall of S. parasitica, chitin is essential for hyphal growth as the inhibition of its biosynthesis leads to highly reduced growth. Here we identified and characterized chitin synthases (CHS) from S. parasitica as potential targets for anti-oomycete drugs. Bioinformatics analyses allowed the identification of six different putative Chs genes in the genome of the pathogen. The total number of genes was confirmed by Southern blot analysis and their expression levels were determined by quantitative PCR. Four of the six Chs genes were expressed in the mycelium, while the two others exhibited undetectable levels of expression. The mycelium was highly sensitive to the addition of nikkomycin Z (NZ) in the culture medium, which led to a decreased amount of chitin in the cell wall by up to 40% in the conditions tested, and to the formation of abnormal branching structures in the hyphae. The presence of NZ increased the expression level of one of the genes, Chs3, suggesting that the corresponding product is compensating the disruption of chitin biosynthesis in the hyphae. In addition, the activity of isolated CHS was strongly inhibited by NZ in vitro. Altogether our data indicate the importance of CHS for the vegetative growth of S. parasitica and demonstrate that these enzymes represent promising targets for the control of diseases caused by oomycetes.

Molecular and physiological characterization of the chitin synthase B gene isolated from Culex pipiens pallens (Diptera: Culicidae)

BackgroundThe growth and development of insects is strictly dependent on the precise regulation of chitin synthase (CHS), which is absent in vertebrates and plants. Therefore, CHS represents an attractive target for insecticides. At present, the research on the CHS gene in mosquitoes, especially its biological functions, remains limited.ResultsThe full-length cDNA of the chitin synthase B gene in Culex pipiens pallens (CpCHSB) was prepared and consists of 5158 nucleotides with an open reading frame (ORF) of 4722 nucleotides encoding a protein of 1573 amino acid residues. Among different tissues, CpCHSB gene is mainly expressed in the midgut tissue with the highest expression in adult mosquitoes. Knockdown of CpCHSB in the larval stage significantly lowered the chitin content (16.5%) decreased body size (reduced by 25.6% in the larval stage and by 25.6% in the adult stage), and diminished reproduction (20%). Injecting siCHSB into adult mosquito mainly decreased reproduction (27%).ConclusionsCpCHSB plays essential roles in growth and development, by severely reducing larval chitin content, midgut permeability, and reducing the number of female mosquito offspring. These results indicate that CHSB may serve as a potential novel target for exploring biosafe insecticides.

The family of amphioxus chitin synthases offers insight into the evolution of chitin formation in chordates

Chitin is a very important and widely-used biopolymer in fungi and lower metazoans, but mysteriously disappears in mammals. Recent studies reveal that at least lower vertebrates have chitin synthases (CS) and use them to synthesize endogenous chitin. Amphioxus, a basal chordate, therefore becomes critical to understand the evolution of CS, as it occupies the transitional position from invertebrates to vertebrates, and is considered as a good proxy to the chordate ancestor.Here, by exploiting multiple genome assemblies, high-depth RNA-seq data and synteny relations, we identify 11–12 CS genes for each amphioxus species. It represents the largest CS gene pool ever found in eukaryotes so far. As comparison, most metazoans have one or two CSs. Amphioxus is the only chordate that has both the very ancient type-I CS family and the more broadly distributed type-II CS family. Specifically, amphioxus has only one type-II CS but 10–11 type-I CSs, which means that amphioxus is the only metazoan with a greatly expanded type-I CS family. Further analysis suggests that the chordate ancestor have at least one type-II CS and an expanded of type-I CS family. We hypothesize that: these ancient CSs are mostly retained in amphioxus; but the whole type-I CS family was lost in urochordates and vertebrates; the type-II CS was later duplicated into two lineages in vertebrates and followed by stochastic losses, till all type-II CSs were eventually lost in birds and mammals. Finally, our expression profiling and preliminary gene knockout analysis suggest that amphioxus CSs could have highly diverse but mildly overlapping functions in various tissues and organs.Taken together, these findings not only provide insights into the evolution of chordate CSs, lay a foundation for further functional study of the chordate CSs. After all, it is mysterious that our chordate ancestor needed so many isoenzymes for chitin formation.

Evaluation of the Expression and Function of the TRE2-like and TRE2 Genes in Ecdysis of Harmonia axyridis.

Harmonia axyridis is an important predatory insect and widely used in biological control of agricultural and forestry pests. Trehalose is directly involved in the energy storage of the H. axyridis and in the oxidative function of various physiological activities thereby providing an energy source for its growth and development. The aim of this study was to explore the potential function of membrane-bound-like trehalase (TRE2-like) and membrane-bound trehalase (TRE2) genes in H. axyridis by RNAi. In addition, the activity of soluble and membrane-bound trehalase and the expression of genes related to trehalose and glycogen metabolism were determined in the larvae injected with dsTRE2-like or dsTRE2. The results showed that wing abnormality and mortality appeared in adults, as well as the activity of soluble trehalase and glycogen contents increased when interfering with TRE2-like gene. However, the activity of membrane-bound trehalase, trehalose and glucose contents in the larvae decreased. The expression of glycogen synthase (GS) and glycogen phosphorylase (GP) genes were decreased after RNAi in the ecdysis stage. The expression of chitin synthase gene A (CHSA), chitin synthase gene B (CHSB), and trehalose-6-phosphate synthase genes (TPS) were decreased significantly after RNAi, especially in the ecdysis stage. These results indicated that RNA interference is capable of knocking down gene expression of TRE2-like and TRE2, thereby disrupting trehalose metabolism which affects the chitin synthesis pathway in turn and also leads to developmental defects, such as wing deformities. This study could provide some theoretical guidance for the function of TRE2 gene in other insects.

First Report of Anthracnose Disease Caused by Colletotrichum truncatum on the Velvetleaf (Abutilon theophrasti) in China

Velvetleaf (Abutilon theophrasti), an annual plant in the family Malvaceae, is an important source of fiber and medicine (Flora of China 2007; Su et al. 2010). Dark brown lesions were observed on the leaves of A. theophrasti in the Herbal Garden of Jilin Agricultural University (125.23°E, 43.47°N), Jilin Province, China, in July 2015 and 2016, respectively. The disease incidence was about 20%. The lesions appeared initially as small, light brown spots on the leaves. These enlarged gradually and coalesced with others to form large spots having irregular edges. Finally, the central portions of the lesions broke open leaving small holes on the leaves. A small piece of tissue was cut from the lesion margin of 30 leaves and then surface sterilized in 75% ethanol for 30 s followed by 1.5% NaClO for 1 min, washed three times with sterilized distilled water, and then dried on sterilized filter paper. Each tissue piece was incubated on potato dextrose agar at 25°C in the dark for 7 days. Colonies formed and were light olive (after 3 days) and then became dark olive (after 7 days). The growth rate (colony radius) ranged from 3.5 to 5.2 mm per day. Conidia were hyaline, aseptate, both ends were bent slightly, and measured 21.9 to 28.0 (25.4 ± 1.4) μm in length and 3.2 to 5.8 (4.3 ± 0.6) μm in width. The hyphae were hyaline, septate, and branched. Appressoria were light to dark brown, solitary, with the outline roundish to ellipsoidal, and 7.3 to 9.8 × 4.8 to 6.3 μm in size. Setae were dark brown, two to five septate, and measured 79.2 to 162.6 μm in length. The morphological characteristics corresponded to Colletotrichum truncatum more clearly and directly (Damm et al. 2009). To further confirm the identity of the pathogen, DNA was extracted from one single-spore isolate, CT12. The following loci were amplified according to Weir et al. (2012): the internal transcribed spacer (ITS) region of ribosomal DNA and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin-2 (TUB2), and chitin synthase (CHS-1) genes. The numbers of base pairs matching were 548/550 for ITS, 225/228 for GAPDH, 241/242 for CHS-1, and 683/683 for TUB2 compared with the sequences of C. truncatum in GenBank (KX621963 for ITS, MK359986 for GAPDH, MK118057 for CHS-1, and MH925229 for TUB2). Maximum likelihood analysis of the combined four gene sequences and reference sequences of Colletotrichum spp. from GenBank and the results of the phylogenetic tree showed that CT12 belonged in C. truncatum with 100% bootstrap support. Sequences of the representative isolate CT12 were deposited in NCBI GenBank (MG993325 [ITS, 558 bp], MH038172 [GAPDH, 225 bp], MH038171 [TUB2, 683 bp], and MH038173 [CHS-1, 242 bp], respectively). Conidial suspension (10⁶ conidia/ml) of CT12 was sprayed on 10 healthy, 3-month-old velvetleaf leaves, and sterilized distilled water was sprayed on 10 healthy 3-month-old velvetleaf leaves as a control. The experiment was conducted three times. All plants were incubated in a controlled incubator with a 12-h photoperiod and 90% relative humidity at 25°C. After 5 days, necrotic lesions occurred on the inoculated leaves but not on the control plants. The same fungus was reisolated from the infected leaf tissues. C. truncatum has been reported to cause anthracnose on soybean, pepper, tomato, eggplant, muskmelon, chickpea, grapes, and other plants (Damm et al. 2009; Diao et al. 2014). To our knowledge, this is the first report of C. truncatum causing anthracnose on velvetleaf in China. The identification of the pathogen will provide a theoretical basis for the identification and control of the disease and reduce economic losses.

The Myosin Motor Domain-Containing Chitin Synthases Are Involved in Cell Wall Integrity and Sensitivity to Antifungal Proteins in Penicillium digitatum.

Penicillium digitatum is the main postharvest pathogen of citrus fruit and is responsible for important economic losses in spite of the massive use of fungicides. The fungal cell wall (CW) and its specific component chitin are potential targets for the development of new antifungal molecules. Among these are the antifungal peptides and proteins that specifically interact with fungal CW. Chitin is synthesized by a complex family of chitin synthases (Chs), classified into up to eight classes within three divisions. Previously, we obtained and characterized a mutant of P. digitatum in the class VII gene (ΔchsVII), which contains a short myosin motor-like domain (MMD). In this report, we extend our previous studies to the characterization of mutants in chsII and in the gene coding for the other MMD-Chs (chsV), and study the role of chitin synthases in the sensitivity of P. digitatum to the self-antifungal protein AfpB, and to AfpA obtained from P. expansum. The ΔchsII mutant showed no significant phenotypic and virulence differences with the wild type strain, except in the production and morphology of the conidia. In contrast, mutants in chsV showed a more dramatic phenotype than the previous ΔchsVII, with reduced growth and conidial production, increased chitin content, changes in mycelial morphology and a decrease in virulence to citrus fruit. Mutants in chsVII were specifically more tolerant than the wild type to nikkomycin Z, an antifungal inhibitor of chitin biosynthesis. Treatment of P. digitatum with its own antifungal protein AfpB resulted in an overall reduction in the expression of the chitin synthase genes. The mutants corresponding to MMD chitin synthases exhibited differential sensitivity to the antifungal proteins AfpA and AfpB, ΔchsVII being more susceptible than its parental strain and ΔchsV being slightly more tolerant despite its reduced growth in liquid broth. Taking these results together, we conclude that the MMD-containing chitin synthases affect cell wall integrity and sensitivity to antifungal proteins in P. digitatum.

Effect of Long-Term Fungicide Applications on Virulence and Diversity of Colletotrichum spp. Associated to Olive Anthracnose.

In this study, the presence and variability of Colletotrichum spp. was evaluated by comparing fungal isolates obtained from olive trees under long-time phytosanitary treatments with trees without any phytosanitary treatments (treated and untreated, respectively). Olive fruits of trees of the highly susceptible ‘Galega vulgar’ cultivar growing in the Alentejo region were used as samples. From the 210 olive trees sampled (half from treated and half from untreated orchards), 125 (59.5%) presented Colletotrichum spp., with a significant lower number of infected trees in treated (39) when compared to untreated orchards (86). The alignment and analysis of beta-tubulin (tub2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), chitin synthase (CHS-1) and histone H3 (HIS-3) gene sequences allowed the identification of all 125 isolates as belonging to the C. acutatum complex. The vast majority of the isolates (124) were identified as C. nymphaeae and one isolate, from an untreated tree, was identified as C. godetiae. Isolates were divided into five different groups: Group A: 39 isolates from treated trees matched in 100% with C. nymphaeae sequences from the database; Group B: 76 isolates from untreated trees matched in 100% with C. nymphaeae sequences from the database; Group C: one isolate from untreated trees presenting a single nucleotidic difference in the HIS-3 sequence; Group D: eight isolates from untreated trees presenting differences in two nucleotides in the tub2 sequences that changed the protein structure, together with differences in two specific nucleotides of the GAPDH sequences; Group E: one isolate, from untreated olive trees, matched 100% with C. godetiae sequences from the database in all genes. Considering the similarities of the sampled areas, our results show that the long-time application of fungicides may have caused a reduction in the number of olive trees infected with Colletotrichum spp. but an increase in the number of fruits positive to Colletotrichum spp. within each tree, which may suggest different degrees of virulence of Colletotrichum isolates from trees growing different management regimes. It is imperative that the fungicides described as causing resistance are applied at appropriate times and intervals, since their efficiency decreases when applied incorrectly and new and more virulent species may arise.

Members of chitin synthase family in Metarhizium acridum differentially affect fungal growth, stress tolerances, cell wall integrity and virulence.

Chitin is an important component of the fungal cell wall with a family of chitin synthases mediating its synthesis. Here, we report on the genetic characterization of the full suite of seven chitin synthases (MaChsI-VII) identified in the insect pathogenic fungus, Metarhizium acridum. Aberrant distribution of chitin was most evident in targeted gene knockouts of MaChsV and MaChsVII. Mutants of MaChsI, MaChsIII, MaChsIV showed delayed conidial germination, whereas ΔMaChsII and ΔMaChsV mutants germinated more rapidly when compared to the wild-type parent. All MaChs genes impacted conidial yield, but differentially affected stress tolerances. Inactivation of MaChsIII, MaChsV, MaChsVII resulted in cell wall fragility, and ΔMaChsV and ΔMaChsVII mutants showed high sensitivity to Congo red and calcofluor white, suggesting that the three genes are required for cell wall integrity. In addition, ΔMaChsIII and ΔMaChsVII mutants showed the highest sensitivities to heat and UV-B stress. Three of seven chitin synthase genes, MaChsIII, MaChsV, MaChsVII, were found to contribute to fungal virulence. Compared with the wild-type strain, ΔMaChsIII and ΔMaChsV mutants were reduced in virulence by topical inoculation, while the ΔMaChsVII mutant showed more severe virulence defects. Inactivation of MaChsIII, MaChsV, or MaChsVII impaired appressorium formation, affected growth of in insecta produced hyphal bodies, and altered the surface properties of conidia and hyphal bodies, resulting in defects in the ability of the mutant strains to evade insect immune responses. These data provide important links between the physiology of the cell wall and the ability of the fungus to parasitize insects and reveal differential functional consequences of the chitin synthase family in M. acridum growth, stress tolerances, cell wall integrity and virulence.

Chitin synthase is involved in vegetative growth, asexual reproduction and pathogenesis of Phytophthora capsici and Phytophthora sojae.

Chitin is a structural and functional component of the fungal cell wall and also serves as a pathogen-associated molecular pattern (PAMP) that triggers the innate immune responses of host plants. However, no or very little chitin is found in the fungus-like oomycetes. In Phytophthora spp., the presence of chitin has not been demonstrated so far, although putative chitin synthase (CHS) genes, which encode the enzymes that synthesize chitin, are present in their genomes. Here, we revealed that chitin is present in the zoospores and released sporangia of Phytophthora, and this is most consistent with the transcriptional pattern of PcCHS in Phytophthora capsici and PsCHS1 in Phytophthora sojae. Disruption of the CHS genes indicated that PcCHS and PsCHS1, but not PsCHS2 (which exhibited very weak transcription), have similar functions involved in mycelial growth, sporangial production, zoospore release and the pathogenesis of P. capsici and P. sojae. We also suggest that chitin in the zoospores of P. capsici can act as a PAMP that is recognized by the chitin receptors AtLYK5 or AtCERK1 of Arabidopsis. These results provide new insights into the biological significance of chitin and CHSs in Phytophthora and help with the identification of potential targets for disease control.

Silencing of the Chitin Synthase Gene Is Lethal to the Asian Citrus Psyllid, Diaphorina citri.

Chitin synthase is a critical enzyme that catalyzes N-acetylglucosamine to form chitin, which plays an important role in the growth and development of insects. In this study, we identified a chitin synthase gene (CHS) with a complete open reading frame (ORF) of 3180 bp from the genome database of Diaphorina citri, encoding a protein of 1059 amino acid residues with the appropriate signature motifs (EDR and QRRRW). Reverse transcription-quantitative PCR (RT-qPCR) analysis suggested that D. citri CHS (DcCHS) was expressed throughout all developmental stages and all tissues. DcCHS had the highest expression level in the integument and fifth-instar nymph stage. Furthermore, the effects of diflubenzuron (DFB) on D. citri mortality and DcCHS expression level were investigated using fifth-instar nymph through leaf dip bioassay, and the results revealed that the nymph exposed to DFB had the highest mortality compared with control group (Triton-100). Silencing of DcCHS by RNA interference resulted in malformed phenotypes and increased mortality with decreased molting rate. In addition, transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH) also revealed corresponding ultrastructural defects. Our results suggest that DcCHS might play an important role in the development of D. citri and can be used as a potential target for psyllid control.

First Report of Colletotrichum liriopes Causing Leaf Spots on Liriope spicata in China

Liriope spicata (Thunb.) Lour. is an evergreen herb, belonging to the genus Liriope, family Liliaceae. As a traditional Chinese medicine, L. spicata is widely cultured in China, Korea, and Japan for ornamental and medicinal purposes. In May 2018, 80 to 90% of L. spicata leaves were observed with leaf spots on the campus of Qingdao Agricultural University. The diseased leaves had oval or irregularly shaped spots with dark brown edges and yellow halos. Some lesions were observed with black acervuli with setae. One fungus was isolated consistently by placing surface-sterilized tissue pieces from the edge of the leaf spots on potato dextrose agar. The resulting colonies were initially whitish and then turned gray after 4 days of incubation at 25°C, and salmon pink spore masses without setae were also observed. Conidia were one-celled (17 to 24 × 2 to 5 μm), hyaline, and falcate. Based on the colony and conidial morphology, the isolate fungus was identified as Colletotrichum liriopes (Damm et al. 2009; Trigiano 2013). The sexual stage and sclerotia formation in the culture were not observed. Sequences of the internal transcribed spacer region of rDNA (GenBank accession no. MK131716), chitin synthase gene (MK144289), β-tubulin gene (MK144290), glyceraldehyde-3-phosphate dehydrogenase gene (MK144291), and calmodulin gene (MK144292) were amplified and analyzed from two single-spore isolates, Ls-CL-01 and Ls-CL-02 (Damm et al. 2009; Weir 2012; White et al. 1990). Because the sequences amplified from Ls-CL-02 were identical to those of Ls-CL-01, only one set of sequences was submitted to GenBank. These sequences had 99 to 100% nucleotide identity to that of C. liriopes (MG543776.1, GU228294.1, KC244160.1, KY995371.1, and HM582017.1, respectively) in GenBank. In addition, MK131716, MK144289, MK144290, and MK144291 had 99% nucleotide identity to that of C. liriopes ex-type CBS 119444 (GU227804, GU228294, GU228098, and GU228196) (Damm et al. 2009). The pathogenicity test was performed using the spray inoculation method. Spores were harvested from 14-day culture plates of Ls-CL-01 and Ls-CL-02 using sterilized distilled water with 0.025% of Tween 20 (Sigma-Aldrich). The spore suspension was adjusted to a final concentration of 5 × 10⁶ spores/ml and was sprayed over five healthy plants, and sterilized water with 0.025% of Tween 20 was used as a control treatment. The inoculated plants were incubated at 25°C and 90% relative humidity with 12-h/12-h dark/light for 3 days and then moved to normal conditions (12-h/12-h dark/light, 20% relative humidity) for 2 days. The experiment was repeated twice. After 5 days of inoculation, approximately 50% of leaves of the inoculated plants (all 15 plants) showed symptoms similar to those previously observed on the original leaves, whereas the control leaves remained asymptomatic. Three isolates were recovered from inoculated leaves. They were identical to isolate Ls-CL-01 in colonial and conidial features. To our knowledge, this is the first report that C. liriopes causes leaf spots on L. spicata. Leaf spots could greatly affect the value of L. spicata; identification of the pathogen provides scientific basis for the disease management.

Effects of RNAi-based silencing of chitin synthase gene on moulting and fecundity in pea aphids (Acyrthosiphon pisum)

The pea aphid, Acyrthosiphon pisum, is an important agricultural pest and an ideal model organism for various studies. Chitin synthase (CHS) catalyses chitin synthesis, a critical structural component of insect exoskeletons. Here, we identified a CHS gene from A. pisum, ApisCHS. The ApisCHS expression profiles showed that ApisCHS was expressed in various developmental stages and in all tested tissues of A. pisum, including the epidermis, embryo, gut and haemolymph. Notably, ApisCHS exhibited peak expression in the middle of each nymphal period and was extremely highly expressed in the epidermis and embryo. RNA interference (RNAi) showed that ~600 ng of dsRNA is an effective dose for gene silencing by injection for dsRNA delivery; moreover, 1200 ng·μL−1 dsRNA induced CHS gene silencing by a plant-mediated feeding approach. A 44.7% mortality rate and a 51.3% moulting rate were observed 72 h after injection of dsApisCHS into fourth-instar nymphs, compared with the levels in the control (injected with dsGFP). Moreover, a longer period was required for nymph development and a 44.2% deformity rate among newborn nymphs was obtained upon ingestion of dsApisCHS. These results suggest that ApisCHS plays a critical role in nymphal growth and embryonic development in pea aphids, and is a potential target for RNAi-based aphid pest control.

Focal distribution of diflubenzuron resistance mutations in Culex pipiens mosquitoes from Northern Italy

Insecticide resistance is a major threat for vector control and prevention of mosquito borne diseases. In the Culex pipiens mosquitoes, resistance against diflubenzuron (DFB) was firstly detected in Ravenna (Emilia-Romagna region, Northern Italy), in 2015. The resistant phenotypes were associated with two mutations, I1043 M and I1043 L, at the amino acid 1043 of the chitin synthase gene. In this study, we monitored the presence, frequency and geographical distribution of the DFB resistant mutations in Cx. pipiens populations from Northern Italy, and in populations from Greece and France. In the Emilia-Romagna region, the resistant mutations were detected in 20 out of the 30 populations analysed, reaching allelic frequencies over 70%. The presence and distribution of the resistance mutations was highly focal, with a clear pattern of increasing resistant allelic frequencies moving from the Western towards the Eastern provinces of Emilia-Romagna. Contrary to Italy, DFB resistant alleles were not detected in the Cx. pipiens mosquitoes sampled from Greece and France. Following statistical, literature and bibliographical database analyses on the history of DFB insecticide use in the study areas, we suggest that the selection pressures from the intense agricultural DFB applications occurring throughout the’ 80-‘90 s against orchard pests, followed, from 2000s onwards by mosquito control DFB applications, may account for the high mutation frequencies observed in the Cx. pipiens populations of the Eastern provinces of Emilia-Romagna. The findings are of major concern for public health in Italy and Europe, as DFB remains a very important insecticide used for controlling arbovirus mosquito vectors, where alternative larvicides are extremely limited.