Subbaratnam Muthukrishnan

Insect chitinases: molecular biology and potential use as biopesticides.

Chitin, an insoluble structural polysaccharide that occurs in the exoskeletal and gut linings of insects, is a metabolic target of selective pest control agents. One potential biopesticide is the insect molting enzyme, chitinase, which degrades chitin to low molecular weight, soluble and insoluble oligosaccharides. For several years, our laboratories have been characterizing this enzyme and its gene. Most recently, we have been developing chitinase for use as a biopesticide to control insect and also fungal pests. Chitinases have been isolated from the tobacco hornworm, Manduca sexta, and several other insect species, and some of their chemical, physical, and kinetic properties have been determined. Also, cDNA and genomic clones for the chitinase from the hornworm have been isolated and characterized. Transgenic plants that express hornworm chitinase constitutively have been generated and found to exhibit host plant resistance. A transformed entomopathogenic virus that produces the enzyme displayed enhanced insecticidal activity. Chitinase also potentiated the efficacy of the toxin from the microbial insecticide, Bacillus thuringiensis. Insect chitinase and its gene are now available for biopesticidal applications in integrated pest management programs. Current knowledge regarding the molecular biology and biopesticidal action of insect and several other types of chitinases is described in this mini-review.

Over-expression of the cloned rice thaumatin-like protein (PR-5) gene in transgenic rice plants enhances environmental friendly resistance to Rhizoctonia solani causing sheath blight disease

Abstract A 1.1-kb DNA fragment containing the coding region of a thaumatin-like protein (TLP-D34), a member of the PR-5 group, was cloned into the rice transformation vector pGL2, under the control of the CaMV 35S promoter. The Indica rice cultivars, ‘Chinsurah Boro II’, ‘IR72’, and ‘IR51500’ were transformed with the tlp gene construct by PEG-mediated direct gene transfer to protoplasts and by biolistic transformation using immature embryos. The presence of the chimeric gene in T0, T1, and T2 transgenic plants was detected by Southern blot analysis. The presence of the expected 23-kDa TLP in transgenic plants was confirmed by Western blot analysis and by staining with Coomassie Brilliant Blue. Bioassays of transgenic plants challenged with the sheath blight pathogen, Rhizoctonia solani, indicated that over-expression of TLP resulted in enhanced resistance compared to control plants.

Sequence of a cDNA and expression of the gene encoding epidermal and gut chitinases of Manduca sexta

Insects use chitinolytic enzymes to digest chitin in the exoskeleton during the molting process. We have isolated and sequenced a chitinase-encoding cDNA from the tobacco hornworm, Manduca sexta, compared its sequence with genes encoding chitinolytic enzymes from other sources, and studied chitinase gene expression and hormonal regulation during the larval-pupal transformation. The insert DNA in this clone is 2452 nucleotides long with an open reading frame of 1662 nucleotides that encodes a protein of 554 amino acids with a molecular weight of 62 kDa. Several regions of the amino acid sequence in this protein are similar to sequences in yeast, cucumber and bacterial endo-beta-N-acetylglucosaminidases. Hybrid-selection of mRNA and in vitro translation yielded an immunoreactive protein with an apparent molecular mass of 75 kDa, which is similar to the size of a chitinase present in pharate pupal molting fluid. Southern blot analysis indicated that one or two genes related to the cDNA clone are encoding chitinases in the Manduca genome. The major tissues expressing chitinase genes were the epidermis and gut with mRNA levels highest on c. days 5-7 during the fifth larval instar. Injection of 20-hydroxyecdysone into ligated fifth instar abdomens caused about a 10-fold increase in mRNA levels in both epidermis and gut, and topical application of the juvenile hormone mimic, fenoxycarb, suppressed the ecdysteroid-induced accumulation of chitinase RNA.

Development of wheat scab symptoms is delayed in transgenic wheat plants that constitutively express a rice thaumatin-like protein gene

Abstract  The possibility of controlling wheat scab (caused by Fusarium graminearum Schw.) was explored by engineering wheat plants for constitutive expression of pathogenesis-related (PR) protein genes. A rice thaumatin-like protein (TLP) gene (tlp) and a rice chitinase gene (chi11) were introduced into the spring wheat cultivar ’Bobwhite’ by co-transformation of the plasmids pGL2ubi-tlp (ubiquitin/tlp//CaMV 35S/hpt) and pAHG11 (CaMV 35S/chi11//ubiquitin/bar). The transformation was by biolistic bombardment. Bialaphos was used as the selection reagent. The integration and expression of the tlp, bar, chi11 and hpt genes were analyzed by Southern, Northern and Western blot analyses. The four transgenes co-segregated in the T1 progeny of the transgenic plant and were localized at the telomeric region of the chromosome 6A long arm by sequential N-banding and fluorescent in situ hybridization (FISH) using pAHG11 or pGL2ubi-tlp as the probes. Only the transgenes tlp and bar, under the control of the ubiquitin promoter-intron, were expressed. No expression of the chi11 and hpt genes, controlled by the CaMV 35S promoter, was detected in T1 plants. After inoculation with conidia of F. graminearum, the symptoms of scab developed significantly slower in transgenic plants of the T1, T2 and T3 generations expressing the tlp gene than in non-transformed control plants. This is the first report of enhanced resistance to F. graminearum in transgenic wheat plants with constitutive expression of TLP.

Enhanced resistance to sheath blight by constitutive expression of infection-related rice chitinase in transgenic elite indica rice cultivars.

Genetic transformation has been attempted for management of rice sheath blight disease, caused by Rhizoctonia solani. We introduced a PR-3 rice chitinase gene (RC7), isolated from R. solani-infected rice plants, into indica rice cultivars IR72, IR64, IR68899B, MH63, and Chinsurah Boro II by the biolistic and PEG-mediated transformation system. Inheritance was studied up to the T(2) generation by Southern blot analysis. Western blot analysis of transgenic plants with polyclonal antibody revealed the presence of chitinase protein with a molecular weight of 35 kDa that reacts with chitinase antibody. The transformants synthesized different levels of chitinase proteins constitutively and progeny from the plants containing the chitinase gene showed different levels of enhanced resistance when challenged with the sheath blight pathogen R. solani.

Insect resistance of transgenic tobacco expressing an insect chitinase gene.

Chitinase expression in the insect gut normally occurs only during moulting, where the chitin of the peritrophic membrane is presumably degraded. Thus, insects feeding on plants that constitutively express an insect chitinase gene might be adversely affected, owing to an inappropriately timed exposure to chitinase. This hypothesis was tested by introducing a cDNA encoding a tobacco hornworm (Manduca sexta) chitinase (EC 3.2.1.14) into tobacco via Agrobacterium tumefaciens-mediated transformation. A truncated but enzymatically active chitinase was present in plants expressing the gene. Segregating progeny of high-expressing plants were compared for their ability to support growth of tobacco budworm (Heliothis virescens) larvae and for feeding damage. Both parameters were significantly reduced when budworms fed on transgenic tobacco plants expressing high levels of the chitinase gene. In contrast, hornworm larvae showed no significant growth reduction when fed on the chitinase-expressing transgenics. However, both budworm and hornworm larvae, when fed on chitinase-expressing transgenic plants coated with sublethal concentrations of a Bacillus thuringiensis toxin, were significantly stunted relative to larvae fed on toxin-treated non-transgenic controls. Foliar damage was also reduced. Plants expressing an insect chitinase gene may have agronomic potential for insect control

Insect chitinase and chitinase-like proteins.

Insect chitinases belong to family 18 glycosylhydrolases that hydrolyze chitin by an endo-type of cleavage while retaining the anomeric β-(1→4) configuration of products. There are multiple genes encoding chitinases and chitinase-like proteins in all insect species studied using bioinformatics searches. These chitinases differ in size, domain organization, physical, chemical and enzymatic properties, and in patterns of their expression during development. There are also differences in tissue specificity of expression. Based on a phylogenetic analysis, insect chitinases and chitinase-like proteins have been classified into several different groups. Results of RNA interference experiments demonstrate that at least some of these chitinases belonging to different groups serve non-redundant functions and are essential for insect survival, molting or development. Chitinases have been utilized for biological control of insect pests on transgenic plants either alone or in combination with other insecticidal proteins. Specific chitinases may prove to be useful as biocontrol agents and/or as vaccines.

7 – Chitin Metabolism in Insects

Publisher Summary This chapter highlights some of the recent and important findings obtained from studies conducted on the synthesis, structure, physical state, modification, organization, and degradation of chitin in insect tissues, as well as the interplay of chitin with chitin-binding proteins, the regulation of genes responsible for chitin metabolism, and, finally, the targeting of chitin metabolism for insect-control purposes. Chitin is the major polysaccharide present in insects and many other invertebrates as well as in several microbes, including fungi. It serves as the skeletal polysaccharide of several animal phyla, such as the Arthropoda, Annelida, Molluska, and Coelenterata. In several groups of fungi, chitin replaces cellulose as the structural polysaccharide. In insects, it is found in the body wall or cuticle, gut lining or peritrophic matrix (PM), salivary gland, trachea, eggshells, and muscle attachment points. In the course of evolution, insects have made excellent use of the rigidity and chemical stability of the polymeric chitin to assemble both hard and soft extracellular structures such as the cuticle (exoskeleton) and PM respectively, both of which enable insects to be protected from the environment while allowing for growth, mobility, respiration, and communication. All of these structures are primarily composites of chitin fibers and proteins with varying degrees of hydration and trace materials distributed along the structures.

Functional specialization among insect chitinase family genes revealed by RNA interference

The biological functions of individual members of the large family of chitinase-like proteins from the red flour beetle, Tribolium castaneum (Tc), were examined by using gene-specific RNAi. One chitinase, TcCHT5, was found to be required for pupal–adult molting only. A lethal phenotype was observed when the transcript level of TcCHT5 was down-regulated by injection of TcCHT5-specific dsRNA into larvae. The larvae had metamorphosed into pupae and then to pharate adults but did not complete adult eclosion. Specific knockdown of transcripts for another chitinase, TcCHT10, which has multiple catalytic domains, prevented embryo hatch, larval molting, pupation, and adult metamorphosis, indicating a vital role for TcCHT10 during each of these processes. A third chitinase-like protein, TcCHT7, was required for abdominal contraction and wing/elytra extension immediately after pupation but was dispensable for larval–larval molting, pupation, and adult eclosion. The wing/elytra abnormalities found in TcCHT7-silenced pupae were also manifest in the ensuing adults. A fourth chitinase-like protein, TcIDGF4, exhibited no chitinolytic activity but contributed to adult eclosion. No phenotypic effects were observed after knockdown of transcripts for several other chitinase-like proteins, including imaginal disk growth factor IDGF2. These data indicate functional specialization among insect chitinase family genes, primarily during the molting process, and provide a biological rationale for the presence of a large assortment of chitinase-like proteins.

Pathogenesis-related proteins and their genes in cereals

Pathogenesis-related proteins (PR-proteins) are induced in plants in response to attack by microbial or insect pests. They have been classified into several groups (PR-1 through PR-14 at present) based on their amino acid sequences and biochemical functions. Many of these proteins that have been purified from infected plants or seed extracts possess antifungal or insecticidal activity. Genes and cDNA clones for all classes of PR-proteins have been isolated from a variety of cereals. Some of these genes/cDNAs have been used to transform cereals. This review presents a summary of the PR-proteins and their genes characterized from rice, wheat, barley, sorghum and maize. Efforts to improve disease or insect resistance of these cereal plants by genetic engineering using genes for PR-proteins also are discussed. In many cases, the expression of the PR-proteins either singly or in combination appears to improve resistance to fungi or insects. In addition, chromosomal location of the PR-protein genes indicates that members of the same family of PR-protein genes or sometimes even several families of PR-protein genes often are clustered in the cereal genome, suggesting coordinate regulation. Some of these PR-protein genes map closely to quantitative traits loci. Some concerns regarding the use of genes encoding PR-proteins for genetic modification of cereals also are addressed.