Chitinase Gene Expression Research Articles

Enhanced extracellular production of laccase in Coprinopsis cinerea by silencing chitinase gene

Laccase, a copper-containing polyphenol oxidase, is an important green biocatalyst. In this study, Laccase Lcc5 was homologous recombinantly expressed in Coprinopsis cinerea and a novel strategy of silencing chitinase gene expression was used to enhance recombinant Lcc5 extracellular yield. Two critical chitinase genes, ChiEn1 and ChiE2, were selected by analyzing the transcriptome data of C. cinerea FA2222, and their silent expression was performed by RNA interference (RNAi). It was found that silencing either ChiEn1 or ChiE2 reduced sporulation and growth rate, and increased cell wall sensitivity, but had no significant effect on mycelial branching. Among them, the extracellular laccase activity of the ChiE2-silenced engineered strain Cclcc5-antiChiE2-5 and the control Cclcc5-13 reached the highest values (38.2 and 25.5 U/mL, respectively) at 250 and 150 rpm agitation speeds, corresponding to productivity of 0.35 and 0.19 U/mL·h, respectively, in a 3-L fermenter culture. Moreover, since Cclcc5-antiChiE2-5 could withstand greater shear forces, its extracellular laccase activity was 2.6-fold higher than that of Cclcc5-13 when the agitation speed was all at 250 rpm. To our knowledge, this is the first report of enhanced recombinant laccase production in C. cinerea by silencing the chitinase gene. This study will pave the way for laccase industrial production and accelerate the development of a C. cinerea high-expression system.Key points• ChiEn1 and ChiE2 are critical chitinase genes in C. cinerea FA2222 genome.• Chitinase gene silencing enhanced the tolerance of C. cinerea to shear forces.• High homologous production of Lcc5 is achieved by fermentation in a 3-L fermenter.

PH/chitinase dual stimuli-responsive essential oil-delivery system based on mesoporous silica nanoparticles for control of rice blast.

Owing to their surface modifiability, smart mesoporous silica nanoparticles (MSNs) can be designed to respond to plant disease-microenvironmental stimuli, thereby achieving on-demand release of active ingredients to control disease by effectively improving citral (CT) stability. A pH/chitinase dual stimuli-responsive essential oil-delivery system (CT@HMS@CH/TA) was successfully fabricated by encapsulating CT in hollow mesoporous silica (HMS), and coating with tannic acid (TA) and chitosan (CH) within HMS by using the layer-by-layer assembly technique (LbL). CT@HMS@CH/TA with an average particle size of 125.12 ± 0.12 nm and a hollow mesoporous nanostructure showed high CT-loading efficiency (16.58% ± 0.17%). The photodegradation rate of CT@HMS@CH/TA under UV irradiation (48 h) was only 15.31%, indicating a 3.34-fold UV stability improvement. CT@HMS@CH/TA exhibited a higher CT release rate in response to acidic pH and the presence of chitinase, simulating the prevailing conditions as Magnaporthe oryzae infection. Furthermore, CT@HMS@CH/TA exhibited better adhesion without affecting normal rice growth, significantly upregulating chitinase gene expression and enhancing chitinase activity on M. oryzae, thus enhancing CT antifungal activity. CT@HMS@CH/TA improved CT stability and showed intelligent, controlled release-performance and higher antifungal efficacy, thus providing a new strategy for efficient application of essential oils for green control of rice blast disease. © 2024 Society of Chemical Industry.

Impact of chitin-derived β-N-acetyl-d-glucosaminyl-(1,4)-d-glucosamine on chitinase upregulation in Shewanella baltica.

The first steps in chitin degradation in marine bacteria involve chitinase, which produces N,N'-diacetylchitobiose (GlcNAc)2 from chitin. Moreover, in Vibrio bacteria, chitinase activity is enhanced by heterodisaccharide β-N-acetyl-d-glucosaminyl-(1,4)-d-glucosamine (GlcNAc-GlcN) produced from (GlcNAc)2 by chitin oligosaccharide deacetylase (COD). However, the role of COD in other marine bacteria, such as Shewanella, remains unexplored. This study investigates GlcNAc-GlcN's impact on chitinase gene expression and enzyme production in S. baltica ATCC BAA-1091, drawing parallels with Vibrio parahaemolyticus RIMD2210633. Using real-time quantitative PCR, the study assesses the upregulation of chitinase gene expression in S. baltica in response to GlcNAc-GlcN, informed by COD's known ability to produce GlcNAc-GlcN from (GlcNAc)2. In Vibrio, GlcNAc-GlcN considerably upregulates chitinase gene expression. This study posits a similar regulatory mechanism in S. baltica, with preliminary investigations indicating COD's capacity to produce GlcNAc-GlcN. This study highlights the importance of exploring GlcNAc-GlcN's regulatory role in chitin metabolism across diverse marine bacteria. The potential induction of chitinase production in S. baltica suggests broader ecological implications. Further research is crucial for a comprehensive understanding of chitin utilization and regulatory pathways in marine bacterial genera.

Identification of the Csr global regulatory system mediated by small RNA decay in Aeromonas salmonicida.

We investigated the presence and functionality of the carbon storage regulator (Csr) system in Aeromonas salmonicida SWSY-1.411. CsrA, an RNA-binding protein, shared 89% amino acid sequence identity with Escherichia coli CsrA. CsrB/C sRNAs exhibited a typical stem-loop structure, with more GGA motifs, which bind CsrA, than E. coli. CsrD had limited sequence identity with E. coli CsrD; however, it contained the conserved GGDEF and EAL domains. Functional analysis in E. coli demonstrated that the Csr system of A. salmonicida influences glycogen biosynthesis, biofilm formation, motility, and stability of both CsrB and CsrC sRNAs. These findings suggest that in A. salmonicida, the Csr system affects phenotypes like its E. coli counterpart. In A. salmonicida, defects in csr homologs affected biofilm formation, motility, and chitinase production. However, glycogen accumulation and protease production were unaffected. The expression of flagellar-related genes and chitinase genes was suppressed in the csrA-deficient A. salmonicida. Northern blot analysis indicated the stabilization of CsrB and CsrC in the csrD-deficient A. salmonicida. Similar to that in E. coli, the Csr system in A. salmonicida comprises the RNA-binding protein CsrA, the sRNAs CsrB and CsrC, and the sRNA decay factor CsrD. This study underscores the conservation and functionality of the Csr system and raises questions about its regulatory targets and mechanisms in A. salmonicida.

The Regulation of Phosphorus Release by Penicillium chrysogenum in Different Phosphate via the TCA Cycle and Mycelial Morphology.

Phosphate-solubilizing fungi (PSF) efficiently dissolve insoluble phosphates through the production of organic acids. This study investigates the mechanisms of organic acid secretion by PSF, specifically Penicillium chrysogenum, under tricalcium phosphate (Ca3(PO4)2, Ca-P) and ferric phosphate (FePO4, Fe-P) conditions. Penicillium chrysogenum exhibited higher phosphorus (P) release efficiency from Ca-P (693.6mg/L) than from Fe-P (162.6mg/L). However, Fe-P significantly enhanced oxalic acid (1193.7mg/L) and citric acid (227.7mg/L) production by Penicillium chrysogenum compared with Ca-P (905.7 and 3.5mg/L, respectively). The presence of Fe-P upregulated the expression of genes and activity of enzymes related to the tricarboxylic acid cycle, including pyruvate dehydrogenase and citrate synthase. Additionally, Fe-P upregulated the expression of chitinase and endoglucanase genes, inducing a transformation of Penicillium chrysogenum mycelial morphology from pellet to filamentous. The filamentous morphology exhibited higher efficiency in oxalic acid secretion and P release from Fe-P and Ca-P. Compared with pellet morphology, filamentous morphology enhanced P release capacity by > 40% and > 18% in Ca-P and Fe-P, respectively. This study explored the strategies employed by PSF to improve the dissolution of different insoluble phosphates.

Cloning and Expression of Class I Chitinase Genes from Four Mangrove Species under Heavy Metal Stress.

Chitinases are believed to act as defense proteins when plants are exposed to heavy metal stress. Typical Class I chitinase genes were cloned from Bruguiera gymnorrhiza, Rhizophora stylosa, Kandelia obovata, and Avicennia marina using the methods of reverse-transcription-polymerase chain reaction and rapid amplification of cDNA ends. All four cDNA sequences of chitinase from the mangrove plants were 1092 bp in length and consisted of an open reading frame of 831 bp, encoding 276 amino acids. However, there were differences in the sequences among the four mangrove species. Four gene proteins have a signal peptide, are located in the vacuole, and belong to the GH19 chitinase family. The sequence of chitinase was highly similar to the protein sequences of Camellia fraternal chitinases. A real-time polymerase chain reaction was used to analyze the chitinase expressions of the above four mangrove species exposed to different concentrations of heavy metal at different times. The gene expression of chitinase was higher in Bruguiera gymnorrhiza leaves than in other mangrove plant species. With an increase in heavy metal stress, the expression level of Bruguiera gymnorrhiza increased continuously. These results suggest that chitinase plays an important role in improving the heavy metal tolerance of mangrove plants.

Expression of Chitinase and shRNA Gene Exhibits Resistance to Fungi and Virus.

With the increasing global population, saving crops from diseases caused by different kinds of bacteria, fungi, viruses, and nematodes is essential. Potato is affected by various diseases, destroying many crops in the field and storage. In this study, we developed potato lines resistant to fungi and viruses, Potato Virus X (PVX) and Potato Virus Y (PVY), by inoculating chitinase for fungi and shRNA designed against the mRNA of the coat protein of PVX and PVY, respectively. The construct was developed using the pCAMBIA2301 vector and transformed into AGB-R (red skin) potato cultivar using Agrobacterium tumefaciens. The crude protein extract of the transgenic potato plant inhibited the growth of Fusarium oxysporum from ~13 to 63%. The detached leaf assay of the transgenic line (SP-21) showed decreased necrotic spots compared to the non-transgenic control when challenged with Fusarium oxysporum. The transgenic line, SP-21, showed maximum knockdown when challenged with PVX and PVY, i.e., 89 and 86%, while transgenic line SP-148 showed 68 and 70% knockdown in the PVX- and PVY-challenged conditions, respectively. It is concluded from this study that the developed transgenic potato cultivar AGB-R showed resistance against fungi and viruses (PVX and PVY).

PpMYB1 and PpNPR1 interact to enhance the resistance of peach fruit to Rhizopus stolonifer infection

MYB transcription factors play important role in stress-resistance of plants. Nevertheless, the function of MYB TFs in peach Rhizopus rot remains poorly understood. Herein, Pichia guilliermondii treatment activated resistance against Rhizopus stolonifer, as illustrated by reductions in the incidence rate and severity of Rhizopus rot disease, increased enzyme activities and gene expression of chitinase (CHI) and β-1,3-glucanase (GLU), and enhancement of energy production by inducing the activities and expression of H+-ATPase and Ca2+-ATPase, succinate dehydrogenase (SDH), and cytochrome c oxidase (CCO). Moreover, an R1-type MYB, PpMYB1, from peach fruit was induced during R. stolonifer infection and in response to P. guilliermondii treatment. PpMYB1 activated the transcription of PpCHI-EP3 and PpGLU-like genes and the energy metabolism-related gene PpH+-ATPase1 by directly targeting the MBS element. Importantly, PpMYB1 interacted with PpNPR1 to form a heterodimer, which was conducive to enhancing the activation of target gene transcription. Collectively, our findings suggest that PpMYB1 cooperates with PpNPR1 to positively regulate disease resistance by activating the disease defense system and energy metabolism in peaches.

Bioinformatics and Expression Analysis of the Chitinase Genes in Strawberry (Fragaria vesca) and Functional Study of FvChi-14.

Plant chitinases (EC 3.2.1.14) are pathogenesis-related (PR) proteins and are well studied in many plant species. However, little is known about the genomic organization and expression of chitinase genes in strawberries (Fragaria vesca). Here, 23 FvChi genes were identified in the genome of strawberry (F. vesca) and divided into GH18 and GH19 subfamilies based on phylogenetic relationships. A detailed bioinformatics analysis of the FvChi genes was performed, including gene physicochemical properties, chromosomal location, exon-intron distribution, domain arrangement, and subcellular localization. Twenty-two FvChi genes showed upregulation after Colletotrichum gloeosporioides infection. Following the exogenous application of SA, FvChi-3, 4, and 5 showed significant changes in expression. The ectopic expression of FvChi-14 in Arabidopsis thaliana increased resistance to C. higginsianum via controlling the SA and JA signaling pathway genes (AtPR1, AtICS1, AtPDF1.2, and AtLOX3). The FvChi-14 protein location was predicted in the cell wall or extracellular matrix. We speculate that FvChi-14 is involved in disease resistance by regulating the SA and JA signaling pathways. The findings of this study provide a theoretical reference for the functional studies of FvChi genes and new candidates for strawberry stress resistance breeding programs.

Chronic toxicity of broflanilide in Daphnia magna: changes in molting, behavior, and gene expression.

Broflanilide is a novel pesticide used in agriculture that binds to unique receptors on pests; however, the widespread use of broflanilide has led to toxicity in Daphnia magna. At present, little information on the potential threats broflanilide imposes on D. magna is available. Therefore, the present study examined the chronic toxicity of broflanilide in D. magna by comparing changes in molting, neurotransmitter function, and behavior. The results showed that broflanilide caused chronic toxicity in D. magna at a concentration of 8.45μg/L, and growth, development, reproduction, and the development of offspring were affected. In addition, broflanilide affected the molting of D. magna by significantly inhibiting the expression of chitinase, ecdysteroid, and related genes. Broflanilide also affected the expression of γ-glutamic acid, glutamine, gamma-aminobutyric acid, 5-hydroxytryptamine, 5-hydroxytryptophan, dopa, and dopamine. Furthermore, the swimming distance and speed of D. magna were reduced. Taken together, the results demonstrate the chronic toxicity and exposure risk of broflanilide in D. magna.

Physiological and Chitinase Gene Expression Responses of Male Zizania latifolia to Ustilago esculenta Infection

This study explored the effects of Ustilago esculenta inoculation on physiological responses (activities of defense and antioxidant enzymes) and chitinase gene expression in male Zizania latifolia “jiaobai” (without U. esculenta infection, with no galls, but normal flowering). Male jiaobai seedlings were injected at the five-leaf stage with U. esculenta suspension, and the impact on transcription of several genes encoding enzymes was examined. Compared with controls, expression of most enzymes was significantly different at 3 or 12 hours postinjection, and most ZlChi genes were involved in the response to U. esculenta inoculation. Fluorescence quantitative polymerase chain reaction results showed that U. esculenta was present in the roots of male jiaobai inoculated with U. esculenta at the shoot tips. Paraffin sections revealed many fungal hyphae in the roots at 15 d after inoculation, but few in controls. The results provide a basis for further study of the responses of male Z. latifolia to U. esculenta infection.

Chitinase (CHI) of Spodoptera frugiperda affects molting development by regulating the metabolism of chitin and trehalose

Chitin is the main component of insect exoskeleton and midgut peritrophic membrane. Insect molting is the result of the balance and coordination of chitin synthesis and degradation in chitin metabolism under the action of hormones. In this study, a 678 bp dsRNA fragment was designed and synthesized according to the known CHI (Chitinase) sequence of Spodoptera frugiperda. It was injected into the larvae to observe the molting and development of S. frugiperda. At the same time, the activities of trehalase and chitinase, the contents of trehalose, chitin and other substances were detected, and the expression of related genes in the chitin synthesis pathway was determined. The results showed that CHI gene was highly expressed at the end of each instar, prepupa and pupal stage before molting; At 12 and 24 h after dsRNA injection of CHI gene of S. frugiperda, the expression of CHI gene decreased significantly, and the chitinase activity decreased significantly from 12 to 48 h. The expression of chitin synthase (CHSB) gene decreased significantly, and the chitin content increased significantly. Some larvae could not molt normally and complete development, leading to certain mortality. Secondly, after RNAi of CHI gene, the content of glucose and glycogen increased first and then decreased, while the content of trehalose decreased significantly or showed a downward trend. The activities of the two types of trehalase and the expression levels of trehalase genes decreased first and then increased, especially the trehalase activities increased significantly at 48 h after dsCHI injection. And trehalose-6-phosphate synthase (TPS), glutamine: fructose-6-phosphate amidotransferase (GFAT), UDP-N-acetylglucosamine pyrophosphorylases (UAP), hexokinase (HK), glucose-6-phosphate isomerase (G6PI) and phosphoacetylglucosamine mutase (PAGM) all decreased significantly at 24 h, and then increased or significantly increased at 48 h. These results indicated that when the expression of chitinase gene of S. frugiperda was inhibited, it affected the degradation of chitin in the old epidermis and the formation of new epidermis, and the content of chitin increased, which led to the failure of larvae to molt normally. Moreover, the chitin synthesis pathway and trehalose metabolism were also regulated. The relevant results provide a theoretical basis for screening target genes and developing green insecticides to control pests by using the chitin metabolism pathway.

Effects of bile acids supplementation in soybean meal‐based diet on growth, cholesterol status, digestibility and moulting‐related gene expression in white shrimp Litopenaeus vannamei

The study evaluated the effects of bile acids (BA) supplementation in soybean meal (SBM)-based diet on growth, cholesterol concentrations, nutrient digestibility and moulting-related gene expression in white shrimp, Litopenaeus vannamei. Control diet with 485.1 g kg-1 SBM was supplemented with 0, 1 and 2 g kg−1 of BA. Together with a reference diet with fish meal, total of four diets were fed to triplicate groups of shrimp (initial weight: 2.20 g) in a recirculation system for 56 days. Weight gain was higher (p < 0.05) in shrimp fed the reference diet than in shrimp fed the control diet. Shrimp fed the reference diet had higher whole body, hepatopancreatic and haemolymph cholesterol concentrations, dry matter, lipid and cholesterol digestibility than other treatments. The expression of the ecdysteroid receptor gene was the highest in shrimp fed the reference diet and the diet with 0.2% BA, followed by shrimp fed the diet with 1 g kg−1 BA and the lowest in shrimp fed the control diet. Shrimp fed the reference diet, and diets with 1 and 2 g kg−1 BA exhibited higher retinoic-X receptor, chitinase and chitin synthease gene expression than shrimp fed the control diet. Haemocyanin gene expression was higher in shrimp fed the reference diet than in shrimp fed the control diet. The results indicated that shrimp fed high SBM diet showed low body cholesterol concentrations, digestibility and moulting-related gene expression for white shrimp. Bile acids supplementation in SBM-based diet can improve moulting-related gene expressions but not for another physiological parameter.

A Comparative Study on Toxicity of Recombinant Chitinase on A Polyphagous Pest, Helicoverpa Armigera

Holometabolous insects complete their life cycle in four stages: egg, larva, pupa and adult. The larval stage is characterized by instar and on an average most larvae has 4- 6 instar stages that occurs between molts. One of the essential enzyme for the process of moulting is chitinase. Expression of chitinase gene takes place when the insect is ready to moult, wherein the old exoskeleton being made of chitin is replaced with the new exoskeleton. Reports indicate that the introduction of the chitinase enzyme at inappropriate time and at inappropriate concentration will prevent the formation of chitin, resulting in no moult, thus growth retardation of the insect and finally death. Insects own chitinase has shown to be a potential insecticide. In the present study, gene encoding for Helicoverpa armigera chitinase was isolated and the sequence was found to be of 1734bp as compared to the reference sequence. The gene was expressed in a prokaryotic and eukaryotic host system. The protein was purified using Ni- NTA column as the protein was tagged with histidine. Chitinase activity was determined and the obtained enzyme was used for toxicity studies against the same insect. Injection assay, topical application and oral ingestion studies showed that Pichia pastoris expressed recombinant chitinase was more effective as compared to the Escherichia coli expressed recombinant chitinase. In case of injection assay, P. pastoris expressed recombinant chitinase recorded 77% mortality for the highest concentration used. Topical application showed 58% and 40% of malformed pupae for Pichia pastoris end E. coli expressed recombinant chitinase. An significant decrease in weight gain was observed on 2nd day for P. pastoris expressed recombinant chitinase and from 7th day for E.coli expressed recombinant chitinase in the oral ingestion studies. Effectiveness of Pichia pastoris expressed recombinant chitinase might be because of post translational modifications in the eukaryotic system.

Identification and Characterization of Arthrobacter nicotinovorans JI39, a Novel Plant Growth-Promoting Rhizobacteria Strain From Panax ginseng.

A bacterial strain JI39 that had plant growth-promoting traits was isolated from the rhizosphere soil of Panax ginseng. It had the ability to produce high indole-3-acetic acid (13.1 μg/ml), phosphate solubilization (164.2 μg/ml), potassium solubilization (16.1 μg/ml), and nitrogen fixation. The strain JI39 was identified to be Arthrobacter nicotinovorans based on morphological, physiological, and biochemical traits and through 16S rDNA sequence analysis. The optimal culture environment for strain growth was 1.0% NaCl, 30°C, pH 6.0, and without UV irradiation. The strain can produce cellulase and protease. The strain JI39 can significantly promote the growth of ginseng. After ginseng seeds were treated with 3 × 108 CFU/ml of JI39 bacterial suspension, the shoot's length was significantly increased by 64.61% after 15 days. Meanwhile, the fresh weight of 2-year-old ginseng roots was significantly increased by 24.70% with a treatment by the 108 CFU/ml bacterial suspension after 150 days in the field. The gene expression of phenylalanine ammonia-lyase (PAL), β-1.3 glucanase (β-1,3-GA), chitinase (CHI), superoxide dismutase (SOD), and peroxidase (POD) of ginseng was upregulated, and it also can improve the soil urease, phosphatase, invertase, and catalase activity. In conclusion, the bacterial strain JI39 could efficiently promote the growth of ginseng and has the potential to be a good microbial fertilizer for ginseng.

Gene Expression in Cucurbita spp. Root and Crown during Phytophthora capsici Infection

Phytophtora capsici causes major diseases in cucurbit crops worldwide. In this study, we inoculated this pathogen into Cucurbita pepo subsp. pepo susceptible MUCU-16 and C. moschata tolerant M63. The gene expression of plant pathogenesis-related proteins chitinase (CpChiIV), lignin-forming peroxidase (CpLPOX), and defensin (CpDEF) and hormone-related enzymes salicylic acid (CpPAL) and ethylene (CpACO) was analyzed for two weeks post-inoculation in root and crown tissues. Differentially expressed genes were found between genotypes, tissues, days post-inoculation, and inoculated/non-inoculated samples. After inoculation, CpPAL and CpChiIV (crown) were downregulated in MUCU-16, while CpLPOX and CpDEF were upregulated in M63. In inoculated samples, higher expression changes were presented on days 10–14 than on day 3 for CpACO, CpLPOX, and CpDEF genes. Overexpression was higher for CpDEF compared to the other tested genes, indicating good suitability as a marker of biotic stress. The overexpression of CpDEF was higher in crown than in roots for both inoculated genotypes. The basal expression of CpPAL and CpDEF was higher in MUCU-16, but after inoculation, CpPAL and CpDEF gene expression were higher in M63. These changes suggest an association between CpDEF upregulation and tolerance, and between CpPAL downregulation and susceptibility.

Lilium regale Wilson WRKY2 Regulates Chitinase Gene Expression During the Response to the Root Rot Pathogen Fusarium oxysporum.

Root rot, mainly caused by Fusarium oxysporum, is the most destructive disease affecting lily (Lilium spp.) production. The WRKY transcription factors (TFs) have important roles during plant immune responses. To clarify the effects of WRKY TFs on plant defense responses to pathogens, a WRKY gene (LrWRKY2) was isolated from Lilium regale Wilson, which is a wild lily species highly resistant to F. oxysporum. The expression of LrWRKY2, which encodes a nuclear protein, is induced by various hormones (methyl jasmonate, ethephon, salicylic acid, and hydrogen peroxide) and by F. oxysporum infection. In this study, LrWRKY2-overexpressing transgenic tobacco plants were more resistant to F. oxysporum than the wild-type plants. Moreover, the expression levels of jasmonic acid biosynthetic pathway-related genes (NtAOC, NtAOS, NtKAT, NtPACX, NtJMT, NtOPR, and NtLOX), pathogenesis-related genes (NtCHI, NtGlu2, and NtPR-1), and antioxidant stress-related superoxide dismutase genes (NtSOD, NtCu-ZnSOD, and MnSOD) were significantly up-regulated in LrWRKY2 transgenic tobacco lines. Additionally, the transient expression of a hairpin RNA targeting LrWRKY2 increased the susceptibility of L. regale scales to F. oxysporum. Furthermore, an F. oxysporum resistance gene (LrCHI2) encoding a chitinase was isolated from L. regale. An electrophoretic mobility shift assay demonstrated that LrWRKY2 can bind to the LrCHI2 promoter containing the W-box element. Yeast one-hybrid assay results suggested that LrWRKY2 can activate LrCHI2 transcription. An examination of transgenic tobacco transformed with LrWRKY2 and the LrCHI2 promoter revealed that LrWRKY2 activates the LrCHI2 promoter. Therefore, in L. regale, LrWRKY2 is an important positive regulator that contributes to plant defense responses to F. oxysporum by modulating LrCHI2 expression.

Transcriptomic profiling of susceptible and resistant flax seedlings after Fusarium oxysporum lini infection.

In this study transcriptome was analyzed on two fibrous varieties of flax: the susceptible Regina and the resistant Nike. The experiment was carried out on 2-week-old seedlings, because in this phase of development flax is the most susceptible to infection. We analyzed the whole seedlings, which allowed us to recognize the systemic response of the plants to the infection. We decided to analyze two time points: 24h and 48h, because our goal was to learn the mechanisms activated in the initial stages of infection, these points were selected based on the previous analysis of chitinase gene expression, whose increase in time of Fusarium oxysporum lini infection has been repeatedly confirmed both in the case of flax and other plant species. The results show that although qualitatively the responses of the two varieties are similar, it is the degree of the response that plays the role in the differences of their resistance to F. oxysporum.

Effect of Extracts of Cupressus macrocarpa and Alpinia officinarum on Expression of Chitinase and FGF Receptor Genes in Spodoptera frugiperda Sf9 Cell Line

Effect of Extracts of Cupressus macrocarpa and Alpinia officinarum on Expression of Chitinase and FGF Receptor Genes in Spodoptera frugiperda Sf9 Cell Line

Expression interference of a number of Heterodera avenae conserved genes perturbs nematode parasitic success in Triticum aestivum

The cereal cyst nematode, Heterodera avenae is distributed worldwide and causes substantial damage in bread wheat, Triticum aestivum. This nematode is extremely difficult to manage because of its prolonged persistence as unhatched eggs encased in cysts. Due to its sustainable and target-specific nature, RNA interference (RNAi)-based strategy has gained unprecedented importance for pest control. To date, RNAi strategy has not been exploited to manage H. avenae in wheat. In the present study, 40 H. avenae target genes with different molecular function were rationally selected for in vitro soaking analysis in order to assess their susceptibility to RNAi. In contrast to target-specific downregulation of 18 genes, 7 genes were upregulated and 15 genes showed unaltered expression (although combinatorial soaking showed some of these genes are RNAi susceptible), suggesting that a few of the target genes were refractory or recalcitrant to RNAi. However, RNAi of 37 of these genes negatively altered nematode behavior in terms of reduced penetration, development and reproduction in wheat. Subsequently, wheat plants were transformed with seven H. avenae target genes (that showed greatest abrogation of nematode parasitic success) for host-induced gene silencing (HIGS) analysis. Transformed plants were molecularly characterized by PCR, RT-qPCR and Southern hybridization. Production of target gene-specific double- and single-stranded RNA (dsRNA/siRNA) was detected in transformed plants. Transgenic expression of galectin, cathepsin L, vap1, serpin, flp12, RanBPM and chitinase genes conferred 33.24–72.4 % reduction in H. avenae multiplication in T1 events with single copy ones exhibiting greatest reduction. A similar degree of resistance observed in T2 plants indicated the consistent HIGS effect in the subsequent generations. Intriguingly, cysts isolated from RNAi plants were of smaller size with translucent cuticle compared to normal size, dark brown control cysts, suggesting H. avenae developmental retardation due to HIGS. Our study reinforces the potential of HIGS to manage nematode problems in crop plant.