Plant protein inhibitors of cell wall degrading enzymes

Plant cell walls, which consist mainly of polysaccharides (i.e. cellulose, hemicelluloses and pectins), play an important role in defending plants against pathogens. Most phytopathogenic microorganisms secrete an array of cell wall degrading enzymes (CWDEs) capable of depolymerizing the polysaccharides in the plant host wall. In response, plants have evolved a diverse battery of defence responses including protein inhibitors of these enzymes. These include inhibitors of pectin degrading enzymes such as polygalacturonases, pectinmethyl esterases and pectin lyases, and hemicellulose degrading enzymes such as endoxylanases and xyloglucan endoglucanases. The discovery of these plant inhibitors and the recent resolution of their three-dimensional structures, free or in complex with their target enzymes, provide new lines of evidence regarding their function and evolution in plant-pathogen interactions.     

Cell elongation and metabolic turnover of the cell wall as affected by auxin and cell wall degrading enzymes

A cell wall fraction (pectic substances) of oat coleoptile segmentsfed with ¹⁴C-glucose contained more radioactivity under theeffect of auxin than did the control. When labeled segmentswere grown for 6 hr in auxin or glucanase solution the labelin the hemicellulose fraction decreased as growth increased.ß-1,3-Glucanase prepared from the culture of a fungus,Sclerotinia libertiana, induces elongation of segments of thepea stem and the oat coleoptile. Traces of cellulase and pectinmethylesterase contaminating the enzyme preparation are notresponsible for the stimulatory effect. Cellulase seemed tobe rather inhibitory and pectin methylesterase showed only aslight effect on coleoptile elongation. A possible relationshipbetween the metabolic turnover of hemicellulosic polysaccharideand cell wall extension is suggested. (Received February 5, 1968; )     

Study of the release of cell wall degrading enzymes during adhesion of Chlamydomonas gametes

A quantitative assay for cell wall release in Chlamydomonas has been used to study the timing of release of cell wall degrading enzyme (lysin) during adhesion. Lysin activity, which shows a broad pH range and requires divalent cations, is released as a pulse within 1–2 min after mixing of mt^? and mt⁺ gametes. Thereafter, there is no further lysin release. Gametes of both mating types release the activity during aggregation with isolated gametic flagella of the opposite mating type, although mt⁺ gametes appear to release more lysin activity than mt^? gametes. Electrophoretic analysis of cell wall proteins before and after lysin degradation indicate that the major wall proteins are unchanged after wall breakdown.     

Regenerated cell wall of carrot protoplasts isolated from suspension-cultured cells

Protoplasts of Daucus carota L. cultured in a synthetic liquid medium resumed cell division after about 4 days of cultivation. During this lag period, nucleic acid and protein showed only slight increases but the protoplasts commenced cell-wall regeneration soon after the removal of lytic enzymes. The originally spherical protoplasts became ellipsoidal before they underwent division. Radioactive glucose and myo-inositol were readily utilized by the protoplasts. Most of the radioactivity, however, appeared in extracellular polysaccharides and only a small portion was deposited in the regenerated wall. The sugar composition of new cell wall, as studies by chemical analysis and incorporation of labelled precursors, was shown to be considerably different from that of normal cell wall.     

Heterogeneity and cell type-specific localization of a cell wall glycoprotein from carrot suspension cells

EP1, an extracellular protein from carrot (Daucus carota) cell suspensions, has been partially characterized by means of an antiserum and a cDNA clone. In both embryo and suspension cultures different molecular mass EP1 proteins were detected, some of which (31, 32, 52, and 54 kilodaltons) were bound to the cell wall and released into the medium, whereas others (49, 60, and 62 kilodaltons) were more firmly bound to the cell wall and could be extracted with a salt solution. Immunoprecipitation of in vitro translation products revealed a single primary translation product of 45 kilodaltons, suggesting that EP1 heterogeneity is due to differential posttranslational modification. In seedlings organ-specific modification of EP1 proteins was observed, a phenomenon which did not persist in suspension cultures initiated from different seedling organs. In culture EP1 proteins were only found to be associated with vacuolated, nonembryogenic cells, and on these cells they were localized in loosely attached, pectin-containing cell wall material. Purified 52/54 kilodaltons EP1 proteins did not alleviate the inhibitory effect of the glycosylation inhibitor tunicamycin on somatic embryogenesis.     

Arsenal of plant cell wall degrading enzymes reflects host preference among plant pathogenic fungi

Background  

The discovery and development of novel plant cell wall degrading enzymes is a key step towards more efficient depolymerization of polysaccharides to fermentable sugars for the production of liquid transportation biofuels and other bioproducts. The industrial fungus Trichoderma reesei is known to be highly cellulolytic and is a major industrial microbial source for commercial cellulases, xylanases and other cell wall degrading enzymes. However, enzyme-prospecting research continues to identify opportunities to enhance the activity of T. reesei enzyme preparations by supplementing with enzymatic diversity from other microbes. The goal of this study was to evaluate the enzymatic potential of a broad range of plant pathogenic and non-pathogenic fungi for their ability to degrade plant biomass and isolated polysaccharides.     

Plasmalemma patch clamp experiments in plant root cells: procedure for fast isolation of protoplasts with minimal exposure to cell wall degrading enzymes

Summary A convenient and rapid isolation procedure for root cell protoplasts suitable for patch clamp experiments, was developed for root cells of tomato (Lycopersicon esculentum) andPlantago species, grown on hydroculture. The procedure is based on a minimal exposure of cells to cell wall degrading enzyme mixtures. After an incubation period of 30 min in a cell wall degrading enzyme mixture all free floating cells were discarded. Subsequently the root material was rinsed and a second group of cells, still present inside the tissue, was freed by application of mechanical pressure. The newly released protoplasts were filtered and collected on the glass bottom of a patch clamp dish. The bathing medium was rinsed extensively removing cellulose fibrils and protoplasts not attached to the glass. Removal of these cellulose fibrils significantly improved the seal success ratio. The isolated protoplasts were suitable for patch clamp experiments in the cell-attached patch, the whole cell and the isolated patch configuration.Abbreviations BSA bovine serum albumin - BTP bis-tris propane - CAP cell-attached patch - OOP outside out patch - PEG polyethylene glycol - WC whole cell     

AepA of Pectobacterium is not involved in the regulation of extracellular plant cell wall degrading enzymes production

Plant cell wall degrading enzymes (PCWDE) are the major virulence determinants in phytopathogenic Pectobacterium, and their production is controlled by many regulatory factors. In this study, we focus on the role of the AepA protein, which was previously described to be a global regulator of PCWDE production in Pectobacterium carotovorum (Murata et al. in Mol Plant Microbe Interact 4:239–246, 1991). Our results show that neither inactivation nor overexpression of aepA affects PCWDE production in either Pectobacterium atrosepticum SCRI1043 or Pectobacterium carotovorum subsp. carotovorum SCC3193. The previously published observation based on the overexpression of aepA could be explained by the presence of the adjacent regulatory rsmB gene in the constructs used. Our database searches indicated that AepA belongs to the YtcJ subfamily of amidohydrolases. YtcJ-like amidohydrolases are present in bacteria, archaea, plants and some fungi. Although AepA has 28% identity with the formamide deformylase NfdA in Arthrobacter pascens F164, AepA was unable to catalyze the degradation of NdfA-specific N-substituted formamides. We conclude that AepA is a putative aminohydrolase not involved in regulation of PCWDE production.     

Callus culture as substrate for the production of specific cell wall degrading enzymes for derived protoplast isolation

Callus culture of spruce (Picea excelsa LINK) appears to be a suitable substrate for the fungusTrichoderma reesei to produce an efficient extracellular lytic system for protoplast isolation. In comparison with Onozuka R-10 cellulase, a yield of protoplasts from the spruce callus 2·5 higher was obtained. Another testea commercial cellulase DK was less efficient. The addition of Macerozyme R–10 significantly enhanced release of protoplasts within all tested enzyme preparations. No difference in the viability of protoplasts has been observed.     

Screening for microorganism with cell wall lytic activity to produce protoplast-forming enzymes

Several different bacteria and fungi capable of degrading yeast cell walls were isolated in the course of a screening programme. One Streptomyces and one Acremonium strain were found to degrade yeast cell walls extremely well. Both isolates produced enzymes in liquid culture that could be used for protoplasting of Sporobolomyces salmonicolor (DSM 70851) and Rhodotorula rubra (DSM 70403). This fact is quite remarkable as, so far, S. salmonicolor could not be protoplasted by commercially available enzymes. Correspondence to: W. Kaul     

Selection of a Streptomyces strain able to produce cell wall degrading enzymes and active against Sclerotinia sclerotiorum

Control of plant pathogen Sclerotinia sclerotiorum is an ongoing challenge because of its wide host range and the persistence of its sclerotia in soil. Fungicides are the most commonly used method to control this fungus but these can have ecotoxicity impacts. Chitinolytic Streptomyces strains isolated from Brazilian tropical soils were capable of inhibiting S. sclerotiorum growth in vitro, offering new possibilities for integrated pest management and biocontrol, with a new approach to dealing with an old problem. Strain Streptomyces sp. 80 was capable of irreversibly inhibiting fungal growth. Compared to other strains, its crude enzymes had the highest chitinolytic levels when measured at 25°C and strongly inhibited sclerotia from S. sclerotiorum. It produced four hydrolytic enzymes involved in fungal cell wall degradation when cultured in presence of the fungal mycelium. The best production, obtained after three days, was 0.75 U/ml for exochitinase, 0.9 U/ml for endochitinase, 0.16 U/ml for glucanase, and 1.78 U/ml for peptidase. Zymogram analysis confirmed two hydrolytic bands of chitinolytic activity with apparent molecular masses of 45.8 and 206.8 kDa. One glucanase activity with an apparent molecular mass of 55 kDa was also recorded, as well as seven bands of peptidase activity with apparent molecular masses ranging from 15.5 to 108.4 kDa. Differential interference contrast microscopy also showed alterations of hyphal morphology after co-culture. Streptomyces sp. 80 seems to be promising as a biocontrol agent against S. sclerotiorum, contributing to the development of new methods for controlling plant diseases and reducing the negative impact of using fungicides.     

Production of fungal cell wall degrading enzymes by a biocontrol strain of Bacillus subtilis AF 1

Fungal cell wall degrading chitinases and glucanases attained significance in agriculture, medicine, and environment management. The present study was conducted to describe the optimum conditions required for the production of beta-1,4-N-acetyl glucosaminidase (NAGase) and beta-1,3-glucanase by a biocontrol strain of Bacillus subtilis AF 1. B. subtilis AF 1 was grown in minimal medium with colloidal chitin (3.0%) and yeast extract (0.3% YE ) and incubated at pH 7.0 and 30 degrees C on constant shaker at 180 rpm for 6 days produced highest amounts of NAGase. Presence of 0.5 mM of phenyl methyl sulfonyl fluoride (PMSF) and 0.04% of Tween 20 further improved the enzyme production. B. subtilis AF 1 grown in minimal medium with laminarin (1%) and yeast extract (0.3%) for 3 days produced maximum amount of beta-1,3-glucanase. These conditions can be further scaled-up for large-scale production of NAGase and beta-1,3-glucanase by B. subtilis AF 1.     

The Rcs phosphorelay modulates the expression of plant cell wall degrading enzymes and virulence in Pectobacterium carotovorum ssp. carotovorum

Production of plant cell wall degrading enzymes, the major virulence factors of soft-rot Pectobacterium species, is controlled by many regulatory factors. Pectobacterium carotovorum ssp. carotovorum SCC3193 encodes an Rcs phosphorelay system that involves two sensor kinases, RcsC(Pcc) and RcsD(Pcc), and a response regulator RcsB(Pcc) as key components of this system, and an additional small lipoprotein RcsF(Pcc). This study indicates that inactivation of rcsC(Pcc), rcsD(Pcc) and rcsB(Pcc) enhances production of virulence factors with the highest effect detected for rcsB(Pcc). Interestingly, mutation of rcsF(Pcc) has no effect on virulence factors synthesis. These results suggest that in SCC3193 a parallel phosphorylation mechanism may activate the RcsB(Pcc) response regulator, which acts as a repressor suppressing the plant cell wall degrading enzyme production. Enhanced production of virulence factors in Rcs mutants is more pronounced when bacteria are growing in the absence of plant signal components.     

余甘子采后软腐病菌的分离鉴定及其细胞壁水解酶活性

【背景】药食同源的余甘子果实在采后贮藏过程中极易软腐变质,严重影响其品质和经济价值。【目的】明确引起余甘子果实软腐病的病原菌种类及其生长特性和产细胞壁水解酶活性,为余甘子采后软腐病的控制及延长其贮藏期奠定基础。【方法】采用组织块分离法从采后发病的余甘子果实分离病原菌,按照科赫法则确定分离菌株的致病性;采用形态学特征结合rDNA-ITS序列分析对病原菌进行鉴定,测定病原菌菌丝生长和产孢特性,检测产胞外细胞壁水解酶活性。【结果】从具有软腐症状的余甘子果实中分离得到32株真菌,其中菌株DQ23是余甘子采后软腐病的致病菌,通过形态特征结合rDNA-ITS序列将其鉴定为Penicillium choerospondiatis。其菌丝在酵母膏葡萄糖琼脂培养基(YDA)上生长最快,在马铃薯蔗糖琼脂培养基(PSA)上产孢最多。该菌能有效利用多种碳、氮源,适宜产孢的碳源为蔗糖、葡萄糖,氮源为蛋白胨、牛肉膏、酵母膏。菌丝生长的最适温度和pH范围分别为25°C和3.0-5.0,产孢的最适温度和pH范围分别为25°C和4.0-7.0。光照均利于菌丝生长和产孢。该菌具有分解果胶、纤维素的能力,无分解蛋白质、鞣质的能力。【结论】Penicillium choerospondiatis是余甘子果实软腐病的病原菌,研究结果为该病害的防控奠定了基础。