Molecular characterization of four chitinase cDNAs obtained fromCladosporium fulvum-infected tomato

Complementary DNA clones encoding acidic and basic isoforms of tomato chitinases were isolated fromCladosporium fulvum-infected leaves. The clones were sequenced and found to encode the 30 kDa basic intracellular and the 26 and 27 kDa acidic extracellular tomato chitinases previously purified (M.H.A.J. Joostenet al., in preparation). A fourth truncated cDNA which appears to encode an extracellular chitinase with 82% amino acid similarity to the 30 kDa intracellular chitinase was also isolated. Characterization of the clones revealed that the 30 kDa basic intracellular protein is a class I chitinase and that the 26 and 27 kDa acidic extracellular proteins which have 85% peptide sequence similarity are class II chitinases. The characterized cDNA clones represent four from a family of at least six tomato chitinases. Southern blot analysis indicated that, with the exception of the 30 kDa basic intracellular chitinase, the tomato chitinases are encoded by one or two genes. Northern blot analysis showed that the mRNA encoding the 26 kDa acidic extracellular chitinase is induced more rapidly during an incompatibleC. fulvum-tomato interaction than during a compatible interaction. This difference in timing of mRNA induction was not observed for the 30 kDa basic intracellular chitinase.     

Transgenic tobacco plants which express thechiA gene fromSerratia marcescens have enhanced tolerance toRhizoctonia solani

We have prepared independent lines of transgenic tobacco plants which express high levels of theSerratia marcescens ChiA protein intracellulary or extracellularly (in glycosylated or unglycosylated forms). We have measured the susceptibility, of these plants toRhizoctonia solani infection in greenhouse trials and in the field. Transgenic tobacco plants which constitutively express theS. marcescens ChiA protein exhibit tolerance to the fungal pathogenR. solani. Disease tolerance is observed in transgenic tobacco plants which express the bacterial chitinase intra-or extracellulary. This is the first report to document disease reduction in the field in transgenic plants engineered for fungal disease tolerance.     

Physiology of microbial degradation of chitin and chitosan

Chitin is produced in enormous quantities in the biosphere, chiefly as the major structural component of most fungi and invertebrates. Its degradation is chiefly by bacteria and fungi, by chitinolysis via chitinases, but also via deacetylation to chitosan, which is hydrolysed by chitosanases. Chitinases and chitosanases have a range of roles in the organisms producing them: autolytic, morphogenetic or nutritional. There are increasing examples of their roles in pathogenesis and symbiosis. A range of chitinase genes have been cloned, and the potential use for genetically manipulated organisms over-producing chitinases is being investigated. Chitinases also have a range of uses in processing chitinous material and producing defined oligosaccharides.     

Fungal Parasites of the Potato Cyst Nematode Globodera rostochiensis: Isolation and Reinfection

Fungal parasitism of eggs of the potato cyst nematode Globodera rostochiensis was < 1, 3, and 17% at three sites in Sweden. The fungi isolated most frequently from infected eggs were a Septocylindrium-like fungus ( 19 %), Exophiala spp. (17 %), and Cylindrocarpon spp. (13 %). Verticillium suchtasporium was isolated from infected eggs at a low frequency (4%). In laboratory experiments V. suchlasporium infected 93% of the eggs within cysts after 10 days on dilute corn meal agar. This species showed chitinase and protease activity. Infection of eggs by the Septocylindrium-like fungus was moderate, whereas Cylindrocarpon destructans and Cladosporium cladosporoides did not infect eggs. No chitinase activity was found in these fungi, but protease activity was recorded in all. Growth of the fungi in cysts did not influence the number of physiologically disordered eggs.     

Inducible Proteins in Citrus Rootstocks with Different Tolerance Towards the Root Rot Pathogen Phytophthora palmivora

Activities of defence‐related proteins (β‐1,3‐glucanases, chitinases and peroxidases) and concentrations of total soluble phenolics were measured in roots and leaves of non‐infected and infected plants to investigate the response of different citrus rootstock genotypes to the root rot pathogen Phytophthora palmivora Butler. Infection with the pathogen increased concentrations of total proteins, total phenolics and β‐1,3‐glucanase activity in roots of all genotypes, and increases were associated with the extent of root mass reductions and thus susceptibility of the plants. Root chitinase and root peroxidase levels were slightly reduced or unaltered upon infection. β‐1,3‐Glucanase activity was also elevated in leaves of infected plants, but increases did not differ between tolerant and susceptible rootstocks. Effects of root infection on leaves were typically the reverse of effects on roots for chitinase‐ and peroxidase levels and more pronounced in susceptible rootstock genotypes. Although differences in enzyme expression were observed between susceptible and tolerant citrus seedlings, effects were usually associated with disease progression, and not with resistance to P. palmivora. It is suggested that increased activities of the proteins and soluble phenolics studied are not implicated in the primary defence to Phytophthora root diseases, but may contribute to the inhibition of the pathogen during infection in tolerant citrus.     

Secondary cell wall formation in Cryptococcus neoformans as a rescue mechanism against acid-induced autolysis

Growth of the opportunistic yeast pathogen Cryptococcus neoformans in a synthetic medium containing yeast nitrogen base and 1.0–3.0% glucose is accompanied by spontaneous acidification of the medium, with its pH decreasing from the initial 5.5 to around 2.5 in the stationary phase. During the transition from the late exponential to the stationary phase of growth, many cells died as a consequence of autolytic erosion of their cell walls. Simultaneously, there was an increase in an ecto-glucanase active towards β-1,3-glucan and having a pH optimum between pH 3.0 and 3.5. As a response to cell wall degradation, some cells developed an unusual survival strategy by forming 'secondary' cell walls underneath the original ones. Electron microscopy revealed that the secondary cell walls were thicker than the primary ones, exposing bundles of polysaccharide microfibrils only partially masked by an amorphous cell wall matrix on their surfaces. The cells bearing secondary cell walls had a three to five times higher content of the alkali-insoluble cell wall polysaccharides glucan and chitin, and their chitin/glucan ratio was about twofold higher than in cells from the logarithmic phase of growth. The cell lysis and the formation of the secondary cell walls could be suppressed by buffering the growth medium between pH 4.5 and 6.5.     

Regulation and characterization of Thermobifida fusca carbohydrate-binding module proteins E7 and E8

E7, a single domain Family 33 cellulose binding module (CBM) protein, and E8, a non-catalytic, three-domain protein consisting of a Family 33 CBM, a FNIII domain, followed by a Family 2 CBM, were cloned, expressed, purified, and characterized. Western blots showed that E7 and E8 were induced and secreted when Thermobifida fusca was grown on cellobiose, Solka floc, switchgrass, or alfalfa as well as on beta-1,3 linked glucose molecules such as laminaribiose or pachyman. E8 bound well to alpha- and beta-chitin and bacterial microcrystalline cellulose (BMCC) at all pHs tested. E7 bound strongly to beta-chitin, less well to alpha-chitin and more weakly to BMCC than E8. Filter paper binding assays showed that E7 was 28% bound, E8 was 39% bound, a purified CBM2 binding domain from Cel6B was 88% bound, and only 5% of the Cel5A catalytic domain was bound. A C-terminal 6xHis tag influenced binding of both E7 and E8 to these substrates. Filter paper activity assays showed enhanced activity of T. fusca cellulases when E7 or E8 was present. This effect was observed at very low concentrations of cellulases or at very long times into the reaction and was mainly independent of the type of cellulase and the number of cellulases in the mixture. E8, and to a lesser extent E7, significantly enhanced the activity of Serratia marscescens Chitinase C on beta-chitin.     

A single histidine residue modulates enzymatic activity in acidic mammalian chitinase

Mammals express two active chitinases, chitotriosidase and AMCase. Only AMCase displays an extremely acidic pH optimum, consistent with its observed presence in the gastro-intestinal tract. A structural model of AMCase reveals the presence of a conserved histidine residue in the active site. Mutational analyses and molecular dynamics simulations show that His187 is responsible for the acidic optimum and suggest pH dependent modulation of the reaction mechanism that is unique to AMCases. Concluding, His187 is a crucial structural component of the active site of AMCase and this unique feature may serve as a lead for the development of specific inhibitors.