Polygalacturonase, Pectate Lyase and Pectin Methylesterase Activity in Pathogenic Strains of Phytophthora capsici Incubated under Different Conditions

Pectolytic enzymes are found mainly in fungi and bacteria. The most widely occurring enzymes are polygalacturonase (PGs), pectin methylesterase (PMEs) and pectate lyase (PLs) produced during the infection process and during culturing. The secretion of these enzymes results in the disorganization of the plant cell walls, which is responsible for the pathogenicity of the pathogens. These enzymes degrade the pectin of plants causing maceration of plant tissues and the enzyme activity increases under favourable environmental conditions. We have found that Phytophthora capsici , a pathogenic oomycete, produces levels of these three enzymes equal to those produced by soft-rotting Erwinia chrysanthemi . The activity of PGs, PLs and PMEs was investigated at the optimum temperature, pH and ionic strength in highly pathogenic P. capsici strains cultivated in two kinds of liquid medium containing either crude pepper extracts plus pectin or pectin as the carbon source. Virulence tests and enzymes activity showed that there was a high correlation between the enzyme activity and the pathogenicity of P. capsici . The effects of different carbon sources on the enzyme activity showed that pepper extract plus pectin was the best source for the carbon source.     

Limitations of In Vitro Strain Screening Methods for the Selection of Sclerotinia spp. as Potential Mycoherbicides against the Perennial Weed Ranunculus acris

Nineteen isolates of Sclerotinia sclerotiorum and three isolates of S. minor were inoculated on to excised tissues and intact plants of Ranunculus acris (giant buttercup), to evaluate their pathogenicity. All isolates proved pathogenic, with S. sclerotiorum being more pathogenic than S. minor on both excised tissues and intact plants. Seven of the S. sclerotiorum isolates were more pathogenic than the others on excised tissues, but no significant differences in pathogenicity were found between any of the isolates when they were inoculated on to intact plants. The results of this study indicate that the excised tissue method cannot be used to predict whole plant mortality, nor, therefore, the mycoherbicide potential of strains of S. sclerotiorum for this perennial weed.     

Physiological and Molecular Features of the Pathosystem Arabidopsis thaliana L.-Sclerotinia sclerotiorum Libert

The fungal pathogen Sclerotinia sclerotiorum Libert causes rot diseases on many crops worldwide and large economic losses occur frequently because of a lack of resistant varieties. The pathogenesis of S. sclerotiorum and the molecular basis of plant responses to the pathogen are poorly understood. In the present investigation, the process of S. sclerotiorum infection in Arabidopsis thaliana L., a plant that is highly susceptible to this fungus, was analysed. In addition, the defense activation in the host was investigated. A convenient inoculation method using millet grain was developed for S. sclerotiorum in Arabidopsis. The fungus rapidly infected the plants, probably through ball- or cushion-like infection structures. Visible symptoms developed within 24 h and plants were killed 72 h after inoculation. Cellulase, the main enzyme that caused host tissues to rot, was secreted by S. sclerotiorum in a pH-dependent manner. Oxalic acid, another pathogenic factor secreted by the fungus, induced necrotic lesions on the leaves, infection with S. sclerotiorum strongly induced the production of the pathogenesis-related （PR） proteins β-1,3-glucanase and chitinase in Arabidopsis. Furthermore, the PR gene PDF. 1 was induced, but not PR1, indicating that the pathogen activated basal defense of jasmonic acid/ethylene dependence, which is consistent with its necrotrophic characteristics. This pathosystem for Arabidopsis-S. sclerotiorum could provide an approach for the analysis of the interactions between S. sclerotiorum and other crops, thereby facilitating genetic manipulation techniques for controlling this pathogen.     

Role of bacterial proteases in pseudomonal and serratial keratitis

Pseudomonas aeruginosa and Serratia marcescens can cause refractory keratitis resulting in corneal perforation and blindness. These bacteria produce various kinds of proteases. In addition to pseudomonal elastase (LasB) and alkaline protease, LasA protease and protease IV have recently been found to be more important virulence factors of P. aeruginosa . S. marcescens produces a cysteine protease in addition to metalloproteases. These bacterial proteases have a number of biological activities, such as degradation of tissue constituents and host defense-oriented proteins, as well as activation of zymogens (Hageman factor, prekallikrein and pro-matrix metalloproteinases) through limited proteolysis. In this article, the properties of these bacterial proteases are reviewed and the pathogenic roles of these proteases in pseudomonal keratitis are discussed.     

pH modulation differs during sunflower cotyledon colonization by the two closely related necrotrophic fungi Botrytis cinerea and Sclerotinia sclerotiorum

During pathogenesis on sunflower cotyledons, Botrytis cinerea and Sclerotinia sclerotiorum show a striking resemblance in symptom development. Based on pH change profiles, the colonization process of both fungi can be divided into two stages. The first stage is associated with a pH decrease, resulting from an accumulation of citric and succinic acids. The second stage is correlated with a pH increase, resulting from an accumulation of ammonia. In this article, we also report that oxalic acid is produced at the late stage of the colonization process and that ammonia accumulation is concomitant with a decrease in free amino acids in decaying tissues. Sclerotinia sclerotiorum produces eight-fold more oxalic acid and two-fold less ammonia than B. cinerea. Consequently, during sunflower cotyledon colonization by B. cinerea, pH dynamics differ significantly from those of S. sclerotiorum. In vitro assays support the in planta results and show that decreases in pH are linked to glucose consumption. At different stages of the colonization process, expression profiles of genes encoding secreted proteases were investigated. This analysis highlights that the expression levels of the B. cinerea protease genes are higher than those of S. sclerotiorum. This work suggests that the overt similarities of S. sclerotiorum and B. cinerea symptom development have probably masked our recognition of the dynamic and potentially different metabolic pathways active during host colonization by these two necrotrophic fungi.     

Investigations on the Activity of Cell Wall-degrading Enzymes in Young Wheat Plants After Infection with Pseudocercosporella herpotrichoides (From) Deighton

In earlier investigations, it has been demonstrated that Pseudocercosporella herpotrichoides (Fron) Deighton is capable of producing pectolytic and cellulolytic enzymes as well as hemicellulases in vitro. The investigation of enzyme activity in extracts from wheat plants infected with P. herpotrichoides (isolates 21e and R6) and from non-infected plants revealed the activity of the following enzymes: pectin methylesterase (PME), polymethylgalacturonase (PMG), pectin lyase (PL), carboxymethylcellulase (CMCase), xylanase and arabanase. Compared to non-infected plants, the enzyme activity in infected plants was considerably higher; in some experiments, only traces of enzyme activity could be found in control plants. The difference in the enzyme activity in infected as compared to non-infected plants was, in most cases, statistically significant, especially beginning at the end of the second week after inoculation.
The enzyme activity depended on the temperature during plant cultivation; with the exception of pectin methylesterase (PME), the activity of all investigated enzymes increased with temperature and the highest activity was found in plants grown at 20°C. The highest PME activity was measured in plants grown at 10°C; the activity of this enzyme was generally lower at 15 and 20°C.     

Serine proteases and metalloproteases associated with pathogenesis but not host specificity in the Entomophthoralean fungus Zoophthora radicans

The protease activity of a Zoophthora radicans strain that was highly infective toward Pieris brassicae (cabbage butterfly) larvae was compared with that of isogenic strains that were adapted to Plutella xylostella (diamondback moth) larvae through serial passage. All strains produced three distinct serine proteases ranging in size from 25 to 37 kDa; however, the original strain from P. brassicae also produced large amounts of an approximately 46 kDa metalloprotease. Subsequently, a cDNA encoding a 43 kDa (mature enzyme) zinc-dependent metalloprotease, ZrMEP1, was isolated from the original fungal strain and most likely corresponds to the 46 kDa protease observed with in-gel assays. ZrMEP1 possessed characteristics of both the fungalysin and thermolysin metalloprotease families found in some pulmonary and dermal pathogens. This is the first report of this type of metalloprotease from an entomo pathogenic fungus. A cDNA encoding a trypsin-like serine protease, ZrSP1, was also identified and was most similar to a serine protease from the plant pathogen Verticillium dahliae. In artificial media, ZrMEP1 and ZrSP1 were found to be differentially responsive to gelatin and catabolite repression in the fungal strains adapted to P. brassicae and P. xylostella, but their expression patterns within infected larvae were the same. It appears that while these proteases likely play a role in the infection process, they may not be major host specificity determinants.     

Interaction of Sclerotinia sclerotiorum with a resistant Brassica napus cultivar: expressed sequence tag analysis identifies genes associated with fungal pathogenesis

Sclerotinia sclerotiorum is a ubiquitous necrotrophic fungal pathogen capable of infecting a wide range of plants. To identify genes involved in fungal development and pathogenesis we generated 2232 expressed sequence tags (ESTs) from two cDNA libraries constructed using either mycelia grown in pectin medium or tissues from infected Brassica napus stems. A total of 774 individual fungal genes were identified of which 39 were represented only among the infected plant EST collection. Annotation of 534 unigenes was possible following the categories applied to Saccharomyces cerevisiae and the Universal Gene Ontology scheme. cDNAs were identified that encoded potential pathogenicity factors including four endopolygalacturonases, two exopolygalacturonases, and several metabolite transporters. The potential role of these genes, as well as those encoding signal transduction factors, in the infection process is discussed.     

Oxalic acid production and its role in pathogenesis of Sclerotinia sclerotiorum

Abstract Sunflower plants were inoculated with a virulent isolate of Sclerotinia sclerotiorum and with the same isolate nutritionally conditioned to produce small amounts of oxalic acid. The preconditioned isolate behaved as hypovirulent. Tomato plants were inoculated with four S. sclerotiorum isolates of increasing virulence. A close correlation among disease severity, accumulation of oxalic acid, decrease in pH and inhibition of polyphenoloxidase in both infected host tissues was demonstrated. Oxalic acid production as an important factor of virulence in S. sclerotiorum is emphasized and its effect on the phenolic metabolism of the host via inhibition of polyphenoloxidase is suggested.     

Characterization of a recombinant granzyme B derivative as a "restriction" protease

Blood coagulation factor Xa (FXa) and Thrombin are well-known serine proteases often used for processing of recombinant fusion proteins, but because they are purified from bovine blood or other animal sources, there is a risk of pathogenic contaminants in the preparation of the proteases. We report here the characterization of a recombinant serine protease produced in Escherichia coli, which can be used as a specific and efficient alternative to FXa and Thrombin as processing protease. This recombinant protease is derived from human granzyme B (GrB). The protease is found to be very stable in general, and it performs very well in the cleavage of several different fusion proteins tested and was even found superior to processing by FXa in two cases.     

Role of proteases in the release of porcine epidemic diarrhea virus from infected cells

Porcine epidemic diarrhea virus (PEDV), a causative agent of pig diarrhea, requires a protease(s) for multicycle replication in cultured cells. However, the potential role of proteases in the infection process remains unclear. In order to explore this, we used two different approaches: we infected either Vero cells in the presence of trypsin or Vero cells that constitutively express the membrane-associated protease TMPRSS2 (Vero/TMPRSS2 cells). We found that PEDV infection was enhanced, and viruses were efficiently released into the culture fluid, from Vero cells infected in the presence of protease, while in cells without protease, the virus grew, but its release into the culture fluid was strongly hampered. Cell-to-cell fusion of PEDV-infected cells and cleavage of the spike (S) protein were observed in cells with protease. When infected Vero cells were cultured for 3 days in the absence of trypsin but were then treated transiently with trypsin, infectious viruses were immediately released from infected cells. In addition, treatment of infected Vero/TMPRSS2 cells with the protease inhibitor leupeptin strongly blocked the release of virus into the culture fluid. Under electron microscopy, PEDV-infected Vero cells, as well as PEDV-infected Vero/TMPRSS2 cells treated with leupeptin, retained huge clusters of virions on their surfaces, while such clusters were rarely seen in the presence of trypsin and the absence of leupeptin in Vero and Vero/TMPRSS2 cells, respectively. Thus, the present study indicates that proteases play an important role in the release of PEDV virions clustered on cells after replication occurs. This unique observation in coronavirus infection suggests that the actions of proteases are reminiscent of that of the influenza virus neuraminidase protein.     

Biological control of Sclerotinia sclerotiorum (Lib.) de Bary, the causal agent of white mold, by Pseudomonas species on canola petals

Sclerotinia sclerotiorum is an important pathogen on canola. Due to the public concern over pesticide use, alternative methods of disease control, such as biological control, should be considered. Several bacterial strains were isolated from canola and soja plants. Inhibition of S. sclerotiorum by bacterial strains in vitro was assayed on PDA medium in dual culture test. Eight Pseudomonas sp. strains (PB-3, PB-4, PB-5, PB-6, PB-7, PB-8, PB-10 and PB-11) caused inhibition zone against 5. sclerotiorum hyphal growth. The biocontrol potential of the bacteria was tested in a plant assay. Disease suppression was investigated using a petal inoculation technique. Canola petals were pretreated with bacteria, and then inoculated with 5. sclerotiorum ascospores 24 h later. Greenhouse experiment showed that application of Pseudomonas sp. strains (1 x 10(8) cfu ml(-1)) effectively suppressed S. sclerotiorum (1 x 10(5) ascospores ml(-1)) on petals and all of them achieved significant (P<0.01) disease suppression. Fourteen days after inoculation, strain PB-3 had 88/7% disease control and strain PB-4 had 69/9% disease control. Result from all studies indicates PB-3 to be effective biocontrol against S. sclerotiorum of canola. PB-3, PB-4, PB-7, PB-8, PB-10 and PB-11 were identified as Pseudomonas fluorescens biovar III. PB-5 and PB-6 was identified as Pseudomonas fluorescens biovar II. Strains PB-3, PB-4, PB-6, PB-10 and PB-11 produced protease and HCN. Strain PB-5 produce protease; no HCN.     

Secretion of pectic isoenzymes by Sclerotinia sclerotiorum

Abstract Cultures of Sclerotinia sclerotiorum grown on different pectin-related polysaccharides (citrus pectin, apple pectin, sodium polygalacturonate), carboxymethylcellulose (CMC) or glucose as the only carbon source were examined daily for polygalacturonase and pectinase activities. Electrophoretic forms of polygalacturonase and pectin methylesterase activities were revealed using analytical IEF and sodium polygalacturonate and citrus pectin as substrates in overlay gels. A sequence in the production of pectic enzymes and isoenzyme synthesis was found in pectic-polymer cultures corresponding to the induction of several isoenzymes. Enzyme activities in glucose media were associated with three polygalacturonase and two pectinmethylesterase isoforms which were produced constitutively. Sodium-dodecyl-sulphate polyacrylamide-gel electrophoresis followed by immunoblotting with polyclonal antibodies against an exo-polymethylgalacturonase and an exo-polygalacturonase revealed that these exo-enzymes were secreted from the beginning of cultivation in the different culture media showing characteristics of constitutive enzymes.     

Relationship between morological variability of Bacillus subtilis R-623 and biosynthesis of hydrolytic enzymes

By synthetic sorbent chromatography the influence of Bacillus subtilis R-623 morphology on the qualitative and quantitative composition of alpha-amylase and proteases was studied. It was found that morphological variants of natural variability of Bac. subtilis R-623, alpha-amylase producer, differed in their cultural, morphological and physiological properties as well as in the amount of hydrolytic enzymes synthesized per unit of the cultural medium. Cells of P variant produced the highest quantity of alpha-amylase (314 U/lm) and cells of P and M variants synthesized the greatest amount of proteases (4.3 and 4.0 U/ml, respectively). Quantitative variations of alpha-amylase and proteases were measured in the cultural medium of morphological variants of Bac. subtilis R-623 during cultivation. Qualitative composition of those enzymes was determined when their content in the cultural medium was at the highest level. R variant synthesized alpha-amylase and protease, P and S variants alpha-amylase and two proteases, and P and S variants alpha-amylase and two proteases, and M variant one protease.     

Identification and characterization of Sclerotinia sclerotiorum NADPH oxidases

Numerous studies have shown both the detrimental and beneficial effects of reactive oxygen species (ROS) in animals, plants, and fungi. These organisms utilize controlled generation of ROS for signaling, pathogenicity, and development. Here, we show that ROS are essential for the pathogenic development of Sclerotinia sclerotiorum, an economically important fungal pathogen with a broad host range. Based on the organism's completed genome sequence, we identified two S. sclerotiorum NADPH oxidases (SsNox1 and SsNox2), which presumably are involved in ROS generation. RNA interference (RNAi) was used to examine the function of SsNox1 and SsNox2. Silencing of SsNox1 expression indicated a central role for this enzyme in both virulence and pathogenic (sclerotial) development, while inactivation of the SsNox2 gene resulted in limited sclerotial development, but the organism remained fully pathogenic. ΔSsnox1 strains had reduced ROS levels, were unable to develop sclerotia, and unexpectedly correlated with significantly reduced oxalate production. These results are in accordance with previous observations indicating that fungal NADPH oxidases are required for pathogenic development and are consistent with the importance of ROS regulation in the successful pathogenesis of S. sclerotiorum.     

Oxalate-degrading bacteria can protect Arabidopsis thaliana and crop plants against botrytis cinerea

Botrytis cinerea and Sclerotinia sclerotiorum secrete oxalic acid as a pathogenicity factor with a broad action. Consequently, it should be possible to interfere with the infection process by degrading oxalic acid during the interaction of these pathogens with their hosts. We have evaluated the potential of oxalate-degrading bacteria to protect plants against pathogenic fungi. Such bacteria were isolated from agricultural soil and selected on agar plates with Ca-oxalate as the sole carbon source. Four strains were retained with a medium-to-strong protective activity on Arabidopsis thaliana leaves against B. cinerea and S. sclerotiorum. They can provide 30 to 70% protection against fungal infection in different pathosystems, including B. cinerea on A. thaliana, cucumber, grapevine, and tomato. The oxalate-degrading bacteria induced only some marker genes for common plant signaling pathways for defenses, but protective effects were slightly reduced in A. thaliana mutants impaired in the ethylene and jasmonic acid signaling pathways. More detailed studies on the protective mechanism were performed in ox-strain B, identified as Cupriavidus campinensis, by analysis of transposon-tagged mutants that have a reduced ability to degrade oxalic acid.     

Analysis of the effects of Perkinsus marinus proteases on plasma proteins of the Eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassostrea gigas).

We employed two in vitro buffer systems to determine the potential pathogenic effects of Perkinsus marinus serine proteases on the plasma proteins of the eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassostrea gigas). Specifically, this study characterized the oyster plasma protein targets of P. marinus proteases. Additionally, protease-specific inhibitory activity was revealed upon comparison of artificial (PBS) and endogenous (plasma-based) diluents employed during protease digestions. It was found that a C. virginica plasma protein of approximately 35 kDa was eliminated when a standard buffer (PBS) was used as a diluent; however, this protein was preserved when a low-molecular-weight, plasma-based, diluent was used. The results strongly indicate that low-molecular-weight inhibitors of P. marinus proteases are present in oyster plasma. A control (nonparasitic) serine protease, alpha-chymotrypsin, was employed to ascertain the specificity of the protease inhibitors. Although alpha-chymotrypsin possesses ample proteolytic activity for C. virginica plasma proteins, the anti-proteases could specifically inhibit only P. marinus proteases. Such specificity of anti-protease activity is not uncommon among low-molecular-weight serine proteases. The hemolymph target protein was isolated by 2D electrophoresis and isoelectrically isolated for further characterization by N-terminal amino acid sequencing.     

Biological control of Sclerotinia sclerotiorum attacking soybean plants. Degradation of the cell walls of this pathogen by Trichoderma harzianum (BAFC 742)

Two experiments of biological control of Sclerotinia sclerotiorum, one in the greenhouse and the other in the field, were carried out with soybean and Trichoderma harzianum as host and antagonist, respectively. Significant control of disease was achieved in both experiments, but there were no significant differences in plant growths. In the greenhouse, the application of T. harzianum as alginate capsules, increased the survival of soybean plants more than 100% with respect to the disease treatment. In the field, T. harzianum treated plants survived 40% more than those from the disease treatment, showing a similar survival level to control plants. Besides, a significant reduction (62.5%) in the number of germinated sclerotia was observed in the Trichoderma treated plot. Chitinase and 1,3-β- glucanase activities were detected when T. harzianum was grown in a medium containing Sclerotinia sclerotiorum cell walls as sole carbon source. In addition, electrophoretic profiles of proteins induced in T. harzianum showed quantitative differences between major bands obtained in the media induced by S. sclerotiorum cell walls and that containing glucose as a sole carbon source. This revised version was published online in June 2006 with corrections to the Cover Date.     

Extracellular proteases of Staphylococcus spp

Bacterial proteases secreted into an infected host may exhibit a wide range of pathogenic potentials. Staphylococci, in particular Staphylococcus aureus, are known to produce several extracellular proteases, including serine-, cysteine- and metalloenzymes. Their insensitivity to most human plasma protease inhibitors and, even more, the ability to inactivate some of these make the proteases potentially harmful. Indeed, several recent studies have shown that staphylococcal proteases are able to interact with the host defense mechanisms and tissue components as well as to modify other pathogen-derived virulence factors. A tight, cell density-dependent control of proteolytic activity expression, similar to that of the well-defined virulence determinants, further suggests the role of staphylococcal proteases in the infection process. Consistently, alterations in coordinated expression of extracellular proteins markedly diminished the virulence. However, despite these data and the fact that a strain deficient in sspABC operon coding for serine (sspA) and cysteine (sspB) proteases was highly attenuated in virulence in the animal infection model, it was impossible to unambiguously demonstrate the importance of any particular protease as a virulence factor. Therefore, it can be assumed that the orchestrated expression and interaction of a variety of extracellular and cell surface proteins rather than any particular one is responsible for the staphylococcal pathogenicity and that the proteases apparently play an important role in this complex process. Such redundant mechanism is very well suited for promoting the survival of staphylococci under diverse environmental conditions encountered in the infected host.     

Adding value to a recalcitrant and problematic waste: the use of dog hair for fungal keratinolytic protease production

Abstract

The demand for keratinolytic proteases has increased in recent years, in view of their applications in the feed, detergent, fertilizer, leather and textile industries. Recently, studies have focussed on exploration of new and inexpensive carbon sources for their production. Among keratin wastes, dog hair presents no utility and is an environmental concern. In this study, we evaluate the feasibility of using white and melanized dog hair (WDH and MDH, respectively) as alternative substrates for protease production by a Fusarium oxysporum strain. The effects of dog hair concentration, cultivation period, and medium pH on alkaline protease production were investigated using a central composite rotary design (CCRD) and response surface methodology (RSM). The optimization process increased protease activity 14.85- and 7.19-fold, using WDH and MDH, respectively. The alkaline proteases produced from WDH and MDH showed distinct biochemical characteristics. To our knowledge, this is the first report on biotechnological use of this problematic, waste residue. Our results open new avenues for conversion of dog hair into other valuable products, especially feed or fertilizer.