Isolation of an osmotic stress- and abscisic acid-induced gene encoding an acidic endochitinase from Lycopersicon chilense

We have identified one osmotic stress- and abscisic acid-responsive member of the endochitinase (EC 3.2.1.14) gene family from leaves of drought-stressed Lycopersicon chilense plants, a natural inhabitant of extremely arid regions in South America. The 966-bp full-length cDNA (designated pcht28) encodes an acidic chitinase precursor with an amino-terminal signal peptide. The mature protein is predicted to have 229 amino acid residues with a relative molecular mass of 24 943 and pI value of 6.2. Sequence analysis revealed that pcht28 has a high degree of homology with class II chitinases (EC 3.2.1.14) from tomato and tobacco. Expression of the pcht28 protein in Escherichia coli verified that it is indeed a chitinase. Northern blot analysis indicated that this gene has evolved a different pattern of expression from that of other family members reported thus far. It is highly induced by both osmotic stress and the plant hormone abscisic acid. Southern blot analysis of genomic DNA suggested that the pcht28-related genes may form a small multigene family in this species. The efficiency of induction of the gene by drought stress, in leaves and stems, is significantly higher in L. chilense than in the cultivated tomato. It is speculated that, besides its general defensive function, the pcht28-encoded chitinase may play a particular role in plant development or in protecting plants from pathogen attack during water stress.     

Chitinase activities are induced in Eucalyptus globulus roots by ectomycorrhizal or pathogenic fungi, during early colonization

A comparative study of root chitinase activities induced by the ectomycorrhizal basidimycete Pisolithus tinctorius Coker and Couch and the pathogenic fungus Phytophthora cinnamomi , has been carried out. Two chitinases were constitutively present in Eucalyptus globulus ssp. bicostata (Maid et al.) Kirkp. roots. When 7-day-old seedlings were challenged with the ectomycorrhizal fungus, root chitinase activity was stimulated already after 6 h, during the very early stages of ectomycorrhizal colonization. Comparing chitinase electrophoretic patterns induced by symbiotic strains more or less compatible with Eucalyptus , a strong stimulation of chitinase activity indicated a successful interaction, which evolved quickly towards root infection and mature mycorrhizae formation. Root chitinase activity remained constant over 7 days during the establishment of the symbiosis. No new chitinase band was induced by the pathogen, when compared with the symbiotic fungi. Chitinase activity was only stimulated quantitatively after pathogenic infection. Root chitinase activities were also stimulated by fungal cell extracts applied in vitro. Such stimulation mimicked precisely the stimulation by living fungi. The intensity of the plant response to fungal extracts was related to fungal strain aggressiveness.     

Eucalyptus root and shoot chitinases, induced following root colonization by pathogenic versus ectomycorrhizal fungi, compared on one- and two-dimensional activity gels

The present work deals with the effect of root fungal colonization on chitinases activities in Eucalyptus seedlings. Plant chitinases indiced during pathogenic infection are thought to be directed against the fungus, but chitinases induced by ectomycorrhizal fungi may contribute to ectomycorrhizal ontogenesis. Plant responses were compared to determine whether plants induce different chitinases activities in contact with symbionts and pathogens, and whether chitinases are induced systemically in both cases. Despite 2-D analysis of Eucalyptus root chitinolytic activities, induced following root colonization by pathogenic or ectomycorrhizal fungi, it was not possible to differentiate between both infections. Moreover, ectomycorrhizal colonization, as pathogenic infections, led to systemic induction of chitinase activities far from the site of inoculation. Contrasting with roots, the chitinase activities induced in shoots were not positively correlated with ectomycorrhizal strain aggressiveness. The differential stimulation of root chitinase activity by aggressive or non-aggressive ectomycorrhizal strains was related to induction of five additional isoforms in response to contact with the most aggressive strains.     

将菜豆几丁酶基因导入苹果砧木的研究

以苹果砧木八楞海棠（Ｍａｌｕｓｍｉｃｒｏｍａｌｕｓ）为实验材料,用微束激光穿刺法将菜豆几丁酶基因导入苹果叶片,成功地获得了转基因植株。实验结果表明,以苗龄２６天的小苗上部刚展开的叶片再生频率可达１００％。经高渗缓冲液预处理后用激光微束穿刺导入外源ＤＮＡ可得到较高的转化频率。在１５０个再生叶片中经卡那霉素筛选得到２５株绿苗,经ＰＣＲ扩增检测,３株呈阳性结果,表明外源几丁酶基因已整合到苹果的基因组ＤＮＡ中。     

Differential induction of apoplastic peroxidase and chitinase activities in susceptible and resistant wheat cultivars by Russian wheat aphid infestation

The intercellular peroxidase and chitinase activities of three wheat cultivars [Triticum aestivum L. cvs `Tugela DN', `Molopo DN' (Gariep) and `Betta DN'] containing the Dn-1 gene for resistance to the Russian wheat aphid (RWA) Diuraphis noxia (Mordvilko) and the corresponding near-isogenic susceptible cultivars (`Tugela', `Molopo' and `Betta') were studied under conditions of infestation and non-infestation. The aim was to gain information on the mechanism of resistance. The resistance response was induced by RWA infestation. Infestation rapidly induced the activities of both enzymes selectively in resistant wheat to levels of magnitudes higher than those in susceptible wheat. The genetic background in which the Dn-1 resistance gene is bred played a role and the level of activity corresponded to the level of resistance. Immunologic studies confirmed that the induction of enzyme activities was due to the induction of higher protein levels. These results indicate that peroxidase and chitinase may have a role in insect resistance. Received: 20 June 1997 / Revision received: 9 April 1998 / Accepted: 5 June 1998     

The role of Nod signal structures in the determination of host specificity in the Rhizobium-legume symbiosis

During the past five years the structure of nodulation signals from more than a dozen different Rhizobium species has been elucidated. In addition, the role of numerous nod genes in the biosynthesis of the lipooligosaccharides has been identified. This review discusses how Nod signal structure is determined by the specificity of the various biosynthetic steps and how this influences variation in host specificity. Until recently, it appeared that the decorations of a common lipochitooligosaccharide core determine the host-specific recognition of the signals, possibly via specific receptors in the host plant cell. A number of recent publications, however, suggest that beyond the interaction of Nod signals with a putative receptor, certain structural features of Nod factors are involved in controlling the concentration of the signals during their uptake by the root tissue.The authors are with the Institut des Sciences Végétales, Centre National de la Recherche Scientifique, Avenue de la Terrasse, F-91198 Gif-sur-Yvette, France; A. Kondorosi is also with the Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences P.O Box 521, H-6701 Szeged, Hungary.     

Regulated inactivation of homologous gene expression in transgenic Nicotiana sylvestris plants containing a defense-related tobacco chitinase gene.

Summary The class I chitinases are vacuolar proteins implicated in the defense of plants against pathogens. Leaves of transgenic Nicotiana sylvestris plants homozygous for a chimeric tobacco (Nicotiana tabacum) chitinase gene with Cauliflower Mosaic Virus (CaMV) 35S RNA expression signals usually accumulate high levels of chitinase relative to comparable leaves of non-transformed plants. Unexpectedly, some transgenic plants accumulated lower levels of chitinase than nontransformed plants. We call this phenomenon silencing. The incidence of silencing depends on the early rearing conditions of the plants. When grown to maturity in a greenhouse, 25% of plants raised as seedlings in closed culture vessels were of the silent type; none of the plants raised from seed in a greenhouse showed this phenotype. Silencing is also developmentally regulated. Plants showed three patterns of chitinase expression: uniformly high levels of expression in different leaves, uniformly low levels of expression in different leaves, and position-dependent silencing in which expression was uniform within individual leaves but varied in different leaves on the same plant. Heritability of the silent phenotype was examined in plants homozygous for the transgene. Some direct descendants exhibited a high-silent-high sequence of activity phenotypes in successive sexual generations, which cannot be explained by simple Mendelian inheritance. Taken together, the results indicate that silencing results from stable but potentially reversible states of gene expression that are not meiotically transmitted. Gene-specific measurements of chitinase and chitinase mRNA showed that silencing results from co-suppression, i.e. the inactivation of both host and transgene expression in trans. The silent state was not correlated with cytosine methylation of the transgene at the five restriction sites investigated.These authors have both made an equal contribution to this work     

Intracellular transport and processing of a tobacco vacuolar β-1,3-glucanase

The class I β-1,3-glucanases are basic, vacuolar enzymes implicated in the defense of plants against pathogen infection. The tobacco (Nicotiana tabacum L.) enzyme is synthesized as a preproprotein with an N-terminal signal peptide for targeting to the lumen of the endoplasmic reticulum and an N-glycosylated C-terminal extension which is lost during protein maturation. The transport and processing of β-1,3-glucanase in cellsuspension cultures of the tobacco cultivar Havana 425 was investigated by pulse-chase labelling and cell fractionation. We verified that mature β-1,3-glucanase is localized in the vacuole of the suspension-cultured cells. Comparison of the time course of processing in homogenates, the soluble fraction, and membrane fractions indicates that proglucanase is transported from the endoplasmic reticulum via the Golgi compartment to the vacuole. Processing to the mature form occurs in the vacuole. Treatment of cells with tunicamycin, which inhibits N-glycosylation, and digestion of the ³⁵S-labelled processing intermediates with endoglycosidase H indicate that β-1,3-glucanase has a single N-glycan attached to the C-terminal extension. Glycosylation is not required for proteolytic processing or correct targeting to the vacuole.     

Chitinase and peroxidase in effective (fix+) and ineffective (fix−) soybean nodules

Chitinase and peroxidase, two enzymes thought to be involved in the defense of plants against pathogens, were measured in soybean (Glycine max L. Merr.) roots and in nodules colonized by Bradyrhizobium japonicum strains differing in their symbiotic potential. Activities of both enzymes were higher in nodules than in roots. In effective, nitrogen-fixing nodules, colonized by wild-type bacteria, chitinase and peroxidase activities had low levels in the central infected zone and were enhanced primarily in the nodule cortex. An ascorbate-specific peroxidase, possibly involved in radical scavenging, had similarly high activities in the infected zone and in the cortex. Ineffective nodules colonized by bacteria unable to fix nitrogen symbiotically showed a similar distribution of chitinase and peroxidase. In another type of ineffective nodule, colonized by a B. japonicum strain eliciting a hypersensitive response, activities of both enzymes were enhanced to a similar degree in the infected zone as well as in the cortex. Tissue prints using a direct assay for peroxidase and an antiserum against bean chitinase corroborated these results. The antiserum against bean chitinase cross-reacted with a nodule protein of M_r 32 000; it inhibited most of the chitinase activity in the nodules but barely affected the chitinase in uninfected roots. It is concluded that proteins characteristic of the defense reaction accumulate in the cortex of nodules independently of their ability to fix nitrogen, and in the entire body of hypersensitively reacting nodules.Abbreviations PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate This work was supported by the Swiss National Science Foundation, Grants 31-25730.88 (to R.B. Mellor and T. Boller) and 31-27923.89 (to A. Wiemken).