WO1994025579A2 - New alkaline serine protease of paecilomyces lilacinus - Google Patents

New alkaline serine protease of paecilomyces lilacinus Download PDF

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WO1994025579A2
WO1994025579A2 PCT/EP1994/001395 EP9401395W WO9425579A2 WO 1994025579 A2 WO1994025579 A2 WO 1994025579A2 EP 9401395 W EP9401395 W EP 9401395W WO 9425579 A2 WO9425579 A2 WO 9425579A2
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protease
gly
ser
ala
thr
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PCT/EP1994/001395
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French (fr)
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WO1994025579A3 (en
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Efje Den Belder
Peter Bonants
Paul Fitters
Cees Waalwijk
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Research Institute For Plant Protection
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Priority to EP94916184A priority Critical patent/EP0700432A1/en
Priority to AU67949/94A priority patent/AU6794994A/en
Publication of WO1994025579A2 publication Critical patent/WO1994025579A2/en
Publication of WO1994025579A3 publication Critical patent/WO1994025579A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins

Definitions

  • nematodes are important parasites. Chemical control of these parasites meet with growing objections because of environmental effects. Alternatives of these control measurements have to be developed. Biological control of nematodes seems to be a good alternative. Many fungi parasitize on plant parasitic nematodes, either by capturing nematodes or by parasitizing the nematode eggs.
  • fungi are capable to penetrate the eggs of nematodes (Bursnall & Tribe (1974), Stirling & Mankau (1979), Chalupova & Lenhart (1984), Kunert et al. (1987), Dackmann et al. (1989) and Gaspard et al. (1990).
  • Those fungi are important objects for research concerning potential use as biological control agents of nematodes.
  • Hyphae of Dactylella oviparasitica grow rapidly through egg-masses of the root-knot nematode Meloidogyne spp. and the fungus penetrated egg-shells (Stirling & Mankau (1979). Dackmann et al.
  • the nematode egg-parasite Paecilomyces lilacinus is able to degrade the egg-shell of the root-knot nematode Meloidogyne hapla (Dunn et al. (1982) and is used as a biological control agent in Peru (Jatala et al. (1980) and in the Philippines (Villanueva & Davide (1984)).
  • the egg-shell of the root-knot nematode M. hapla consits for at least 40 % of protein (Bird & McClure (1976), Bird (1976)).
  • the outer layer of the egg-shell consists of vitellin, also a protein, and is therefore the first substrate for the fungus.
  • the invention relates to
  • a process for the preparation of the protease characterized under 1 which comprises cultivation of Paecilomyces lilacinus and isolation of said protease.
  • the invention further relates to the gene for a protease, having the DNA- sequence shown in the sequence protocol 1 and for a gene coding for an enzyme having the same action and whose amino acid sequence is coded by the DNA sequence shown in sequence protocol 1 and derived from that sequence by addition, deletion or exchange of nucleotides.
  • the serine protease from Paecilomyces lilacinus exhibits an enzymatic activity towards surface structures of plant parasitic nematodes, preferably the egg shell, especially vitellin.
  • isolation and purification can be carried out as described in the materials and methods and in the examples.
  • the enzyme preparation can be characterized by a molecular weight of 20,000 to 200,000, preferably 25,000 to 100,000, especially 30,000 to 50,000 Dalton.
  • the enzyme can be characterized furthermore by an isoelectric point which is at a pH of 8 to 12, preferably 9 to 11, especially 9,5 to 12.
  • the pH optimum of the enzyme product is in the range of 7 to 12, preferably 8 to 11, especially 9,5 to 11.
  • the reaction temperature can range between 40 and 80 °C, preferably between 50 and 70, especially between 55 and 65 °C.
  • transaminase directly or indirectly for the control of plant parasitic nematodes.
  • the invention additionally relates to plasmids containing a gene of this type, and to microorganisms, in particular E.coli and fungi, containing a plasmid of this type.
  • Another object of the invention is to provide a new process for controlling the action of nematodes on plants and plant cells.
  • the gene can be used to control plant parasitic cyst, root-knot and lesion nematodes, especially root-knot nematodes.
  • the gene is used to control the growth of Meloidogyne hapla and Meloidogyne incognita belonging to the Heteroderidae. This family includes also the potato cyst nematode and the sugar beet cyst nematode.
  • Another object is to provide DNA fragments which comprise DNA sequences capable of protecting plants and plant cells, when incorporated and expressed therein against the action of plant parasitic nematodes.
  • the preparation of transformed plants comprises the following steps:
  • the promotor contains the DNA sequence which is necessary for the inition of transcription. Further downstream, i.e. following the promotor, is the so-called 5' non translated region which is also involved in the initiation of transcription. In most cases the promotor will be located at the 5' end of the gene, but it can also vary in its position.
  • the coding region is followed further downstream by the so-called 3' untranslated region. This region does contain signals which cause the termination of transcription and in eucargotic cells an additional signal that causes the polyadenylation of the transcribed RNA.
  • the above constructed DNA sequence are examples for chimeric genes.
  • the DNA sequences which regulate the expression may be derived from different sources, e.g. plant, virus or bacterial genes which are active in plants.
  • promotors which cause konstitutiv or inducible expression.
  • Inducible promotors may be expressable depending on the development of the cell or tissue specific.
  • Preferred promotors include the Cauliflower Mosaic Virus (CAMV) 35S promotor, the nopaline synthase promotor, the octopine synthase promotor and the ubiquitin promotor.
  • the chimeric genes may also include leader sequences and signal sequences.
  • the DNA construct can be introduced into the plant cell using different techniques that are described in the art. These methods include direct gene transfer (EP 0 164 575), ballistic particle, microinjection and Agrobacterium mediated transformation (EP 0 116 718, Hoekema and An et al.).
  • the selection of plant cells which have been transformed is enabled by the use of a selectable marker gene which is also transferred.
  • the expression of the marker gene confers a phenotypic trait that enables the selection. Examples for such genes are those coding for antibiotica or herbicide resistance, e.g. neomycin or phosphinothricin resistance.
  • Plants which can be protected may be either monocotyledons or dicotyledons.
  • Examples of families that are of special interest are Solanaceae and Brassicaceae. Examples of species of commercial interest that can be protected include:
  • tobacco Nicotiana tabacum L. tomato, Lycopersicon esculentum Mill, potato, Solanum tuberosum L, petunia, Petunia hybrida (Solanaceae) Canola/Rapeseed, Brassica napus L, cabbage, broccoli, kale etc., Brassica oleracea L, mustards Brassica juncea L, - Brassica nigra L, and Sinapis alba L (Brassicaceae),
  • Beta vulgaris (Chenopodiaceae)
  • the gene prevents diseases of plants such as potato, tomato, wheat, cabbage and Chinese cabbage.
  • Figure 1 Protease activity of culture filtrates of Paecilomyces lilacinus (10 8 conidia) in 10 ml liquid medium (NM: minimal medium, CFM: corn flour medium) to which different substrates were added: E: 2,500,000 Meloidogyne hapla eggs for MME and 760,000 M. hapla eggs for CFME, N: nitrogen (NaN0 3 (0.2 %) + asparagine (0.2 %)) and NG: nitrogen (NaN0 3 (0.2 %) + asparagine (0.2 %)) + glucose (2.0 %).
  • NM minimal medium
  • CFM corn flour medium
  • Figure 2 Protease activity / ⁇ g protein of culture filtrates of Paecilomyces lilacinus (mycelium after 6 days of growth in 250 ml MNNG) in 250 ml liquid medium (MN: minimal medium + nitrogen (NaN0 3 (0.2 %) + asparagine (0.2 %)) to which chitin was added as substrate. Glucose was added daily.
  • CHIT colloidal chitin (0.2 % or 1.0 % (w/v),
  • GLUC glucose (0.3 % (w/v) / day).
  • Figure 3 SDS-PAGE patterns of filtrates of 4 days old cultures of Paecilomyces lilacinus in minimal medium to which different substrates were added: Lane NG: Nitrogen + Glucose; Lane V: Vitellin; Lane C: Chitin; Lane E: Eggs; Lane M: Markers, Molecular weight of markers are indicated at the left side.
  • Figure 4 Protease activity of fractions of Bactracin-Sepharose column to which concentrated culture filtrate of Paecilomyces lilacinus, grown in minimal medium with chitin as substrate, was added. Elution was started at fraction 7. Procedure was described under Materials & Methods.
  • Figure 5 Protease activity of purified protease (1.875 ⁇ g) of Paecilomyces lilacinus at different pH. Measurements were performed in triplicate. Protease was purified as described under Materials & Methods from culture filtrate of P. lilacinus in minimal medium with vitellin as substrate.
  • Figure 6 Protease activity of purified protease (1.875 ⁇ g) of Paecilomyces lilacinus at different temperatures. Measurements were performed in triplicate. Protease was purified as described under Materials & Methods from culture filtrate of P. lilacinus in minimal medium with vitellin as substrate.
  • Figure 7 Protease activity of purified protease (0.47 ⁇ g) of Paecilomyces lilacinus after incubation for 40 minutes at 4 °C with different inhibitors, SH-reducing agents and metal ions. Measurements were performed in triplicate. Final inhibitor concentration was 1 mM. Protease was purified as described under Materials & Methods from culture filtrate of P.
  • Protease- inhibitors were respectively none, PMSF, 1,10-phenanthroline, Pepstatine, E64, DTT, Cysteine, CaCI 2 , MgCI 2 and EDTA.
  • Figure 8 Sequence of primer A and primer B. I: Inosine residue; Y: Cytidine or Thymidine; R: Adenine or Guanine.
  • Figure 9 Restriction map of pSP3, pSP4, pSP3-2. The probable location of the 240-bp PCR-generated fragment is shown. E, EcoRI; H, Hindi; H?, Hindi, location uncertain; N, Ncol; S, Smal; X, Xhol. No Avail, BamHI, Notl, Nrul, Pstl, Sad, Sacll sites, no internal EcoRI and Xhol sites.
  • Table 1 Protease activity and protein content of filtrates of 4 days old culture of Paecilomyces lilacinus (2x10 8 conidia) in 200 ml liquid medium (MM: minimal medium) to which different substrates were added: MMC : colloidal chitin (0.2 % (w/v)),
  • MMC colloidal chitin (0.2 % (w/v)),
  • MME 450,000 Meloidogyne hapla, eggs
  • Paecilomyces lilacinus (Thorn.) Samson (CBS 143.75), obtained from the CBS (Central Bureau of Fungal Cultures) in Baarn (The Netherlands), was routinely maintained (once a month) on Potato Dextrose Agar (PDA; difco laboratories) in 90 mm petridishes at 25 °C.
  • a conidial suspension was obtained by adding 5 ml of sterilized water to a PDA petridish containing sporulating mycelium and scraping the surface with a glass rod.
  • Liquid cultures were obtained by inoculating conidia of the fungus to minimal salt medium or corn flour medium supplemented with the substrate.
  • the minimal salt medium (MM) consisted of 4.56 gr I ⁇ HPO ⁇ 2.77 gr KH 2 HP0 4 , 0.5 gr MgS0 4 . 7H 2 0 and 0.5 gr KCI / liter pH 6.0.
  • the corn flour medium (CFM) was prepared by steaping 40 gr of grinded corn flour in 1 I demineralized water for 1 hour at 55 °C. Next the mixture was filtered over Whatman no 1 filter and the filtrate was used after autoclavation (20 minutes 120 °C).
  • the following substrates were added alone or in combination when required: vitellin (0.2 % (w/v); Sigma), collodial chitin (0.2 % and 1.0 % (w/v); prepared according to Lingappa & Lockwood (1962) using chitin from Sigma), root-knot nematode eggs ( > 400.000; isolated as described below), nitrogen (N) (as 2.0 gr asparagine + 2.0 gr NaN0 3 /l) and glucose (G) (2.0 % (w/v).
  • mycelium of Paecilomyces lilacinus was used as inoculum.
  • Mycelium was obtained by centrifuging a 6 day old culture of conidia of P. lilacinus in MMNG for 45 minutes at 9000 g. Cultures were grown in a shaking waterbath for several days at 30 °C and 125 strokes per minute. Culture filtrates were obtained by centrifuging cultures for 45 min at 9000 g. Supernatants were concentrated using an Amicon (YM-10 filter) and clarified through a 0.22 ⁇ m-filter (Schleicher & Schuell). Pellets were freeze dried and the weigth of the mycelium was measured.
  • Escherichia coli cells were grown in LB broth (1 % bacto tryptone, 0.5 % yeast extract, 0.5 % Sodium chloride), when necessary 100 ⁇ g/ml ampicillin was added.
  • E. coli strain PLK-F'(mrcA-, mrcB-, recA-, Tet R ) was used for the preparation of plating cells.
  • E. coli strain InVaF' competent cells were purchased from Invitrogen (San Diego) and used as carrier of recombinant plasmids.
  • a spore suspension of Paecilomyces lilacinus was obtained by adding 5 ml of sterilized water to a PDA plate containing sporulating P. lilacinus mycelium and scraping the surface with a glass rod.
  • Liquid cultures for genomic DNA isolations were grown in potato dextrose broth (PDB; Difco).
  • the liquid induction medium (IM) used for the isolation of the mRNA fraction contained 0.5 gr/L KCI, 0.5 gr/L MgS0 4 , 1.36 gr/L KH 2 P0 4 , 2.28 gr/L K 2 HP0 4 and 4x10 8 nematode eggs/L as the sole carbon and nitrogen source.
  • As an inoculum 4x10 10 spores/L was used. 3.2.2 Culture of nematode
  • the root-knot nematode Meloidogyne hapla Chitwood obtained from the Centre for Plant Breeding and Reprodcution Research (CPRO-DLO) in Wageningen, was continiously maintained in a greenhouse on tomato plants (Lyopersicon esculentum cv. Moneymaker) for 8 hr at 15 °C and 16 hr at 20 °C per day with a relative humidity of > 65%. Artificial lighting was supplied to give 16 hours daylength. The plants are grown in sterilised silver sand and nutrients were supplied according to Steiner.
  • the Northern root-knot Meloidogyne hapla was reared on tomato plants (Lycopersicon esculentum cv. Moneymaker) in a greenhouse.
  • Six week-old plantlets were inoculated by placing 2000 nematode eggs in the vicinity of the stem.
  • Eight to ten weeks after inoculation nematode eggs were collected by vigorously shaking the nematode-infected roots in 1 % (v/w) hypochlorite for 10 min.
  • the liberated eggs were collected by centrifugation (2x10 3 rpm, 10 min), and loaded onto a continous sucrose gradient, prepared by freezing and slowly thawing a 40 % (w/v) sucrose solution. The gradient was spun (2x10 3 rpm, 10 min) and the eggs were collected, washed several times with sterilized, distilled water, and counted.
  • Protease activity was determined by a modified procedure of Rinderknecht et al. (1968). Ten mg of Hide Powder Azure (Sigma) was incubated in 50 mM Sodium acetate buffer pH 6.0 with the sample in a final volume of 3 ml in small glass containers. Incubation was at 37 °C in a shaking waterbath (110 strokes/min) till the solution turned blue (between 5 and 30 minutes). Following incubation the samples were put on ice and centrifuged in 1.5 ml Eppendorf cups for 5 minutes at 14.000 g. The absorbance of the supernatant was measured at 595 nm on a Uvikon 940 spectrophotometer. Protease activity of the sample was measured in duplo, averaged and expressed as A 5g5 ml *1 hr "1 after correction for the blank (Milli Q water).
  • Protease activity on gelatine agar plates was determined by incubating conidia of P. lilacinus on 1.2 % (w/v) NM agar at 25 °C containing 0.2 % (w/v) gelatin. Halo's indicating extracellular proteolytic activity were visualized by staining with Coomassie Brilliant Blue.
  • Protein quantification of the samples were determined according the Bradford (1976), using BSA (bovine serum albumine) as the standard.
  • SDS-PAGE was performed by the method of Laemmli (1970). Culture filtrate samples were precipitated with trichloroacetic acid, washed with ice-cold aceton, dried, suspended in sample-buffer (62.5 mM Tris/HCI pH 6.8, 10 % glycerol, 2 % SDS, 10 mM DTT, 0.1 % BPB) and boiled for 5 minutes. Native PAGE in the presence of gelatin was performed according to Heussen & Dowdle (1980).
  • the concentrated culture filtrate was dialyzed overnight at 4 °C against equilibration buffer (100 mM Ammoniumacetate, 10 mM CaCI 2 , pH 6.5) and applied to an equilibrated 2 ml Bacitracin-Sepharose column.
  • the Bacitracin-Sepharose was made as described by Stepanov & Rudenskaya (1983). After washing the column with 100 mM Ammoniumacetate buffer pH 6.5, the bound protease was eluted with elution buffer (100 mM Ammoniumacetate, 1 M NaCI, 25 % isopropanol pH 6.5). Fractions of 1 ml were collected and tested for proteolytic activity. Positive fractions were pooled and dialyzed agianst 100 nM Ammonium acetate pH 6.5.
  • - Optimum temperature For the determination of the optimum temperature for protease activity the proteolytic activity measurements were performed as described above in triplicate at different temperatures.
  • - Isoelectric point For the determination of the isoelectric point the protease was applied to a Mono P chromatofocusing column (HR 5/5 from Pharmacia) at pH 10.2 (25 mM 2-amino-2-methyl-1-propanol/HCI). With an FPLC (Fast Protein Liquid Chromatography) system gradient elution was performed with Polybuffer (1 :10) pH 8.0.
  • Iso-electrofocusing was employed with the Phastsystem of Pharmacia using lEF-gels with a pH-gradient from 3 to 10 according to the instructions of the manufactory.
  • protease inhibitors Several protease inhibitors, SH reducing agents and metal ions were tested on the proteolytic activity of the protease: PMSF, E64, 1.10- phenantroline, pepstatine (Sigma); EDTA, cystein MgCI 2 and CaCI 2 (Merck); DTT (BioRad).
  • the purified enzyme was incubated for 40 minutes at 4 °C with inhibitor. After incubation the protease-activity was determined as previously described. Final inhibitor concentration was 1 mM. Protease activity in the absence of inhibitor was expressed as 100 %.
  • Root-knot nematode eggs were incubated at room temperature with the purified protease in 1.5 ml Eppendorf cups in 100 mM Potassium phosphate buffer pH 7.0 with continously shaking.
  • Vitellin (1 mg) was incubated with the purified protease for 16 hours at 37 °C in 1.5 ml Eppendorf cups in 100 mM Sodiumacetate buffer pH 6.0 (final volume was 1 ml). After incubation the samples were centrifuged (1 minute 14000 g) and absorption of the supernatant was mesaured at 280 nm on an Uvikon 940 spectrophotometer.
  • P. lilacinus was grown for 2-4 days in PDB at 30 °C and 150 rpm.
  • the mycelium was collected by filtration through myracloth (Calbiochem Corporation, La Jolla).
  • the mycelium was frozen in liquid nitrogen and grinded to a fine powder in a mortar.
  • 5 ml of extraction buffer 0.1 M NaCI, 10 mM Tris-HCI (pH 7.5), 1 mM EDTA, 1 % SDS
  • An equal volume of phenol/chloroform (1 :1) was added and the phases were vigourously mixed.
  • RNAse was added to a final concentration of 10 ⁇ g/ml and the solution was incubated at 37 °C for 30 min. The solution was extracted once with phenol/chloroform (1 :1), once with chloroform and then precipitated with ethanol. 3.2.8 Isolation of mRNA from P. lilacinus
  • P. lilacinus was grown for 48 hours in MM at 30 °C and 150 rpm.
  • the mycelium was collected by filtration through myracloth.
  • the mycelium was frozen in liquid nitrogen and grinded to a fine powder in a mortar.
  • 5 ml of extraction buffer 0.1 M NaCI, 10 mM Tris-HCI (pH 7.5), 1 mM EDTA, 1 % SDS
  • An equal volume of phenol/chloroform (1:1) was added and the phases were vigourously mixed.
  • the PCR reaction mixture of 100 ⁇ L contained 50 mM KCI, 10 mM Tric/HCI (pH 8.3), 0.5-2 mM MgCI 2 , 100 ⁇ M of each dNTP, 100 pmol of oligonucleotide A and oligonucleotide B (see below), 400 ng of genomic DNA from P. lilacinus, and 2.5 units of AmpliTaq DNA polymerase (Perkin Elmer) mineral oil was added to prevent evaporation.
  • Each of the 35 amplification cycles included a denaturation step at 94 °C for 1 min, an annealing step at 42 °C to 60 °C for 2 min, and a chain elongation step at 72 °C for 3 min.
  • the amplification reaction was preceded by a denaturation step at 94 ° for 0.5 min, and the elongation step of the last cycle was extended to 5 min.
  • 3.2.10 Screening of the cDNA library
  • the library was plated at a density of 10.000 plaques/plate on PLK-F' cells. After overnight incubation at 37 °C, duplicate nitrocellulose filters or each plate were prepared according to Maniatis et al (15).
  • the filters were baked at 80 °C for 2 hours and incubated in prehybridisation solution (6x SSC, 5x Denhardt's solution, 0.1 % SDS, 100 ⁇ g/mL denatured salmon sperm DNA) at 65 °C for 4 hours.
  • prehybridisation solution (6x SSC, 5x Denhardt's solution, 0.1 % SDS, 100 ⁇ g/mL denatured salmon sperm DNA
  • a radioactive probe 1 ⁇ L of the PCR reaction product was used.
  • the labelling reaction mixture was identical to the PCR reaction mixture, except that the dATP was substituted for 5 ⁇ L alpha- 32 P-dATP (3000 Ci/mmol, 10 mCi/ml).
  • 7 amplification cycles were performed identical to the cycling conditions used to obtain the DNA fragment.
  • the probe was separated from the free nucleotides using a Sephahdex G-50 spin column. After denaturation (10 min, 100 °C), the probe was added to prehybridisation solution and the filters were hybridized for 16 hours.
  • the filters were washed twice 2x SSC/0.1 SDS for 30 min at 65 °C, and once with 0.2x SSC/0.1 SDS for 30 min at 65 °C.
  • X-ray film (Fuji RX) was exposed to the filters for 16 hours and an autoradiograph was obtained. Positives were rescreened by the same procedure until pure. After in vivo excision of the plasmids according to the manual supplied by the manufacturer (Stratagene), they were subjected to restriction enzyme analysis.
  • the insert of pSP3 was partially sequenced using the Taq dye primer cycle sequencing kit (Applied Biosystems, Foster City) and an Biorad R370 automated sequencer, and the T7 sequencing kit and the Automated Laser Fluorescent DNA Sequencer of Pharmacia. 3.3 Working Examples
  • Paecilomyces lilacinus grown for 3 days at 25 °C on solid agar containing gelatin showed halo's after staining with Coomassie Brilliant Blue proving extracellular production of proteases.
  • the production of extracellular protease by the fungus in liquid minimal salt medium (MM) and in liquid corn flour medium (CFM), to which several substrates were added was studied.
  • Protease activity of the culture filtrate was monitored at daily intervals following inoculation of the medium with conidia of the fungus.
  • Figure 1 shows the protease activities of the culture filtrate with eggs (E) as substrate and with nitrogen and glucose (NG) as control on several days after inoculation.
  • protease activity was seen with vitellin as substrate.
  • Specific protease activity protease activity/ ⁇ g protein was highest in cultures containing eggs as substrate.
  • glucose concentrations were determined in the culture filtrate, of the fungus in MNNG ( Figure 1).
  • Glucose is a well known repressor of the induction of many enzymes.
  • glucose was added at a concentration of 0.3 % each day to MNNC. In this experiment mycelium of P. lilacinus was used as inoculum.
  • Figure 2 shows that the induction of protease activity was repressed by glucose. Adding conidia or mycelium (harvested after incubating the same amount of conidia of P. lilacinus as inoculum in MNNG for 6 days) resulted in no differences in induction of the proteolytic activity.
  • the SDS-PAGE patterns of the culture filtrates showed many proteins produced in the different cultures ( Figure 3).
  • the protein pattern of the culture filtrate with vitellin as substrate looked very similar to the one of the culture filtrate with nematode eggs as substrate (lane 3 and 5 respectively).
  • To identify the nature of the protease the proteolytic activity in the culture filtrate was inhibited with several protease inhibitors. Inhibition of the proteolytic activity in the culture filtrate with PMSF (a serine protease inhibitor) showed that most of the protease activity present in the medium was inhibited suggesting a serine protease being involved as the most secreted protease.
  • Figure 4 shows the proteolytic activity of fractions of the Bacitracin-Sepharose column to which concentrated culture filtrate of the fungus (grown for 4 days in MM with chitin a substrate) was applied. After washing and elution the protease activity was predominantly found in fractions 8 and 9. Integration of the densitogram of fraction 8 revealed that one protein was present for more than 85 %. It was concluded that this was the serine protease. The same protein was purified from culture filtrates of P. lilacinus grown with eggs and vittelin as substrate. All substrates induced the serine protease.
  • the molecular weight of the serine protease of Paecilomyces lilacinus using molecular weigth markers was 33.5 kDa. Since the protease did not bind to the Mono P column at pH 10.2 the isoelectric point should even be higher. Isoelectric focussing using the Phastsystem of Pharmacia and pH 3-10 gels showed that the protease focussed at pH 10.
  • the optimum pH and temperature for proteolytic activity was obtained by performing protease activity measurements at different pH and temperature respectively.
  • Figure 5 shows an optimum pH of 10.3 after fitting of the protease activity curve.
  • Figure 6 shows the temperature activity profile for the P. lilacinus protease.
  • Optimum temperature for the proteolytic activity was about 60 °C.
  • FIG. 7 shows the effect of several protease inhibitors, some SH- reducing agents and some metal ions on the activity of the purified protease.
  • PMSF inhibited the activity for 100 %. Consequently the protease is of the serine protease class. All other protease inhibitors tested did not effect the proteolytic activity of the enzyme significantly.
  • DTT and cystein did not influence its activity also suggesting no SH-groups being involved in the active center of the enzyme.
  • Ca + + - and Mg + + - ions lowered the protease activity slightly.
  • EDTA on the other hand enhanced its activity suggesting a negative influence of metal ions present in the assay.
  • the purified protease was capable of degrading insoluble vitellin and produced halo's on MM agar plates containing gelatin.
  • Psp P. lilacinus serine protease
  • the 16 amino acids of the N-terminus of Psp were determined to be Ala-Tyr-Thr- Gln-Gln-Pro-Gly-Ala-Pro-(His?/Cys?/Trp?)-Gly-Leu-Gly-Arg-lle-(Ser).
  • Comparison of this sequence to other amino acid sequences of fungal subtilisin proteases, as compiled by Tatsumi et al., showed that the C-terminal part of the 15-amino acid stretch contained a conserved region, whereas the N-terminal part showed little homology.
  • the first oligonucleotide was based on this stretch of 9 amino acids to avoid cross- reactions with other subtilisin-like proteases that P. lilacinus may produce (figure 8).
  • Panel A Panel B.
  • Panel A Comparison of the N-terminal sequence of Psp to the N-termini of other subtilisin-like protease.
  • A Aspergillus oryzae Alp (Tatsumi et al.); B, Saccharomyces cerevisiae protease B (Moehle et al.); C, Yarrowia lipolytica alkaline protease (Davidow); D, Tritirachium album, proteinase K (Jany et al.); E, Paecilomyces lilacinus Psp.
  • Panel B Comparison of the amino acid sequences surrounding the active site His residue (see also figure 8: indicated by *).
  • A-D as in panel A; E, hypthetical sequence used for the design of oligonucleotide B.
  • inosine residues were used where four-base wobble occured. Degeneracy was allowed at sites where two-base wobble occured.
  • Primer A 5' GCITAYACICARCARCCIGGIGCICC 3' Primer B: 5' GTICCIGCRCARRGIGTICCRTGICCRTTICC 3 *
  • thermo-stable serine protease of Tritirachium album Limber (Samal et al. (1990)); an alkaline protease of Aspergillus fumigeius (Jaton-Ogay et al. (1992)); an basic proteinase of Trichoderma harzianum (Geremia et al. (1993)); an alkaline protease of Acremonium chysogenium (Isogai et al. (1991)); aqualysin I of Thermus aquaticus (Terada et al.
  • protease B of Saccharomyces cerevisiae (Moehle et al. (19987)) and an alkaline extracellular protease of Yarrowia lipolytica (Davidow et al. (1987)).
  • the temperature of the annealing step of the cycles was varied from 42 °C to 60 °C, resulting in multiple bands in all experiments.
  • the annealing temperature was set at 60 °C and the MgCI 2 - concentration was optimized.
  • MgCI 2 -concentration of 0.5 mM a single DNA band was seen on an agarose gel.
  • the size of the band was estimated to be 240 basepairs. Although several other bands were seen at higher MgCI 2 - concentrations, the 240-bp band was always the most predominant.
  • the PCR reaction product containing the single visible 240-bp DNA band was used a radioactive labelled probe and hybridised to duplicate nitrocellulose filters containing 100.000 plaques in total.
  • the resulting autoradiographs showed both strong and weak hybridisation signals.
  • a restriction map was made of the 7 plasmids, and based on these data they could be classified in two categories.
  • the first category consisting of 4 plasmids, contained an insert of 1400 base pairs.
  • the second category consisting of 3 plasmids, contained an insert of 1200 bp.
  • One of these plasmids was named pSP4. Based on restriction patterns, we concluded that the insert of pSP4 is a shorter version of pSP3 (figure 9).
  • pSP3-2 a subclone, pSP3-2, was created by digesting pSP3 with Smal and religate the larger fragment containing the vector and 0.5 kb of the insert.
  • pSP3 was partially sequenced. It was possible to identify the region containing oligonucleotide A (figure 9). This region is located 250 bp downstream of the EcoRI cloning site. Furthermore, a 3'poly(A) sequence was found.
  • oligonucleotide A The region just downstream of oligonucleotide A is reasonably consistent with the known N-terminal amino acid sequence of Psp, which leads us to believe that pSP3 encodes the Psp protease of P. lilacinus.
  • the length of the insert of pSP3 upstream of the oligonucleotide A sequence suggests that this is a full length or nearly full length cDNA.
  • the whole mature protein sequence is included (see SEQ ID NO 1 and 2, the protein sequence starts at amino acid No. 84 with Ala-Tyr-Thr, see also 3.3.4). The start codon and part of the leader sequence are therefore missing. 4. References.
  • tylenchid (Nematode) egg shell structure, composition and permeability. Parasitology 72, 19-28.
  • BRADFORD M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254.
  • DAVIDE R.G. & BATINO, E. (1985).
  • JATON-OGAY G., SUTER, M., CRAMERI, R., FALCHETTO, R., FAITH, A. & MONOD, M.
  • LAEMMLI U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685.
  • Protease B of the lysosomelike vacuole of the yeast Saccharomyces cerevisiae is homologous to the subtilisin family of serine proteases. Mol. Cell. Biol. 7: 4390- 4399.
  • Cuticle-degrading enzymes of entomopathogenic fungi Cuticle degradation in vitro by enzymes from entomopathogens. Journal of Invertebrate Pathology 47, 167-177.
  • TERADA I., KWON, S.-T., MiYATA, Y., MATSUZAWA, H. & OTHA, T. (1990).
  • VILLANUEVA LM. & Davide, R.G. (1984). Evaluation of several isolates of soil fungi for biological control of root-knot nematodes. Philippine Agriculture 67, 361- 371.
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Paecilomyces lilacinus
  • CTATGTCATC GACACTGGCG TCGAGGCTTC GCACCCCGAG TTCGAGGGCC GCGCCAGCCA 420
  • GGTCCTCGAC AACTCGGGCT CCGGCTCATA CTCGGGCATC ATCTCCGGTA TGGACTTTGC 600 CGTTCAGGAC TCCAAGTCGC GCAGCTGCCC CAAGGGTGTC GTCGCCAATA TGTCTCTGGG 660
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Paecilomyces lilacinus
  • Arg lie Ser His Arg Ser Lys Gly Ser Thr Thr Tyr Glu Tyr Asp Thr 100 105 110
  • ORGANISM Saccharomyces cerevisiae
  • ORGANISM Yarrowia lipolytica
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ORGANISM Paecilomyces lilacinus
  • ORGANISM Saccharomyces cerevisiae
  • ORGANISM Yarrowia lipolytica
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ORGANISM Paecilomyces lilacinus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Paecilomyces lilacinus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Paecilomyces lilacinus

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Abstract

The invention relates to a protease from Paecilomyces lilacinus, a process for the preparation of the protease, which comprises cultivation of Paecilomyces lilacinus and isolation of said protease. More generally the invention relates to the use of the protease for the control of plant parasitic nematodes. The invention further relates to the gene for this protease.

Description

New alkaline serine protease of Paecilomyces lilacinus
1. Introduction
In many crops nematodes are important parasites. Chemical control of these parasites meet with growing objections because of environmental effects. Alternatives of these control measurements have to be developed. Biological control of nematodes seems to be a good alternative. Many fungi parasitize on plant parasitic nematodes, either by capturing nematodes or by parasitizing the nematode eggs.
Several fungi are capable to penetrate the eggs of nematodes (Bursnall & Tribe (1974), Stirling & Mankau (1979), Chalupova & Lenhart (1984), Kunert et al. (1987), Dackmann et al. (1989) and Gaspard et al. (1990). Those fungi are important objects for research concerning potential use as biological control agents of nematodes. Hyphae of Dactylella oviparasitica grow rapidly through egg-masses of the root-knot nematode Meloidogyne spp. and the fungus penetrated egg-shells (Stirling & Mankau (1979). Dackmann et al. (1989) investigated fungal egg-parasites isolated from eggs of the cyst nematode Heterodera avenae with respect to their ability to infect cyst nematode eggs of H. schachtii in vitro. Of these Verticillium suchlasporium appeared to be the most effective parasite. Gaspard et al. (1990) and de Leij & Kerry (1991) reported on Verticillium chlamydosporium as control agent for Meloidogyne spp. Also the nematode egg-parasitic fungus Paecilomyces lilacinus (Thorn) Samson has been studied for its ability to control root-knot nematodes (Jatala et al. (1979, 1980), Dunn et al. (1982), Villanueva & Davide (1984), Davide & Batino (1985), Culbreath et al (1986), Dube & Smart (1987), Cabanillas & Barker (1989), Cabanillas et al. (1989), Gaspard et al. (1990).
The mechanism of the infection process of egg-parasitic fungi could be mechanical and/or enzymatic in nature. Little is known about the role of enzymes involved. To date few attempts have been made to determine the effects of purified fungal enzymes on the egg-shell of the root-knot nematode. Research on enzymes of nematode egg-parasitic fungi was done in liquid culture of Dactylella oviparasitica supplemented with colloidal chitin. Stirling & Mankau (1979) found chitinase activity and suggested a role for this enzyme in penetrating the egg-shell, which consists partly of chitin. If any other enzyme was involved was unclear. Dackmann et al.
(1989) showed a correlation between infection of egg-parasites of cyst nematode eggs Heterodera schachtii in vitro and lytic enzyme activity.
Especially chitinase and protease activity seemed to be involved. Lopez-Llorca
(1990) purified an extracellular protease from the fungal parasite of eggs of cyst nematodes, Verticillium suchlasporium, and proved that this protease was capable of degrading a protein from the egg-shell of cyst nematodes. Lopez-Llorca & Robertson (1992) immunolocalized cytochemically the presence of this protease in infected nematode eggs and supported therefore the role for this enzyme in the pathogenicity of the fungus to nematode eggs.
Den Belder et al. (1993) showed SEM pictures of root-knot nematode eggs infected by P. lilacinus suggesting clearly the involvement of enzymes.
The products of enzymes and especially proteases of several organisms, which are able to degrade a certain kind of shell, for example insect cuticles or fungal cell walls, were studied (Gabriel (1968), Leopoled & Samsinakova (1970), Samsinakova et al. (1971), Latge (1974), Kucera (1980), Smith et al. (1981), St. Leger et al. (1986a, 1986b, 1986c, 1987a, 1991), Geremia et al. (1991). St. Leger and co- workers (1987a) showed that the proteases, produced in situ during penetration of Calliphora vomitoria and Manduca sexta cuticles by hyphae of the entomopathogenic fungus Metarhizium anisopliae, were the same as those produced in culture media. Production of the protease without differentation of infection structures of M. anisopliae can occur rapidly by nutrient deprivation alone (St. Leger et al. (1988). The proteases of two entomopathogenic fungi, M. anisopliae and Beauveria bassiana have been purified (St. Leger et al. (1986a, 1987b), Bidochka & Khachatourians (1987). Gold-labelled antiserum was used to demonstrate that a culticle-degrading protease (Pr1) is produced by M. anisopliae during penetration of host (Manduca sexta) procuticle (Goettel et al. (1989)).
The nematode egg-parasite Paecilomyces lilacinus, is able to degrade the egg-shell of the root-knot nematode Meloidogyne hapla (Dunn et al. (1982) and is used as a biological control agent in Peru (Jatala et al. (1980) and in the Philippines (Villanueva & Davide (1984)). The egg-shell of the root-knot nematode M. hapla consits for at least 40 % of protein (Bird & McClure (1976), Bird (1976)). The outer layer of the egg-shell consists of vitellin, also a protein, and is therefore the first substrate for the fungus.
2. Description of the invention
The invention relates to
1. A alkaline serine protease from Paecilomyces lilacinus having
- a molecular weight of 20,00 to 200,000 Dalton
- an isoelectric point at a pH between 9 and 12,
- a pH optimum in a range from 9 and 12 and
- an enzymatic activity towards surface structures of plant parasitic nematodes.
2. A process for the preparation of the protease characterized under 1 , which comprises cultivation of Paecilomyces lilacinus and isolation of said protease.
3. The use of the protease characterized under 1 for the control of plant parasitic nematodes.
4. The invention further relates to the gene for a protease, having the DNA- sequence shown in the sequence protocol 1 and for a gene coding for an enzyme having the same action and whose amino acid sequence is coded by the DNA sequence shown in sequence protocol 1 and derived from that sequence by addition, deletion or exchange of nucleotides.
The serine protease from Paecilomyces lilacinus exhibits an enzymatic activity towards surface structures of plant parasitic nematodes, preferably the egg shell, especially vitellin.
The investigation revealed that the enzyme is astonishingly active at high temperatures. This feature can be used to distinguish and separate the enzyme from other proteins by thermal denaturation.
The isolation and purification can be carried out as described in the materials and methods and in the examples.
The enzyme preparation can be characterized by a molecular weight of 20,000 to 200,000, preferably 25,000 to 100,000, especially 30,000 to 50,000 Dalton.
The enzyme can be characterized furthermore by an isoelectric point which is at a pH of 8 to 12, preferably 9 to 11, especially 9,5 to 12.
The pH optimum of the enzyme product is in the range of 7 to 12, preferably 8 to 11, especially 9,5 to 11.
The reaction temperature can range between 40 and 80 °C, preferably between 50 and 70, especially between 55 and 65 °C.
It is possible according to the invention to use that transaminase directly or indirectly for the control of plant parasitic nematodes.
The invention additionally relates to plasmids containing a gene of this type, and to microorganisms, in particular E.coli and fungi, containing a plasmid of this type. By taking advantage of genetic engineering the gene responsible for the production of the serine protease can be transferred to plant cells, which can be regenerated to whole plants.
Therefore another object of the invention is to provide a new process for controlling the action of nematodes on plants and plant cells. The gene can be used to control plant parasitic cyst, root-knot and lesion nematodes, especially root-knot nematodes. In a preferred embodiment the gene is used to control the growth of Meloidogyne hapla and Meloidogyne incognita belonging to the Heteroderidae. This family includes also the potato cyst nematode and the sugar beet cyst nematode.
Another object is to provide DNA fragments which comprise DNA sequences capable of protecting plants and plant cells, when incorporated and expressed therein against the action of plant parasitic nematodes.
The preparation of transformed plants comprises the following steps:
ligating the coding region of the serine protease to a promotor and a terminator which are active in plant cells, transferring and integrating said contructed DNA sequence in the genome of a plant cell, regenerating whole plants from transformed cells.
The promotor contains the DNA sequence which is necessary for the inition of transcription. Further downstream, i.e. following the promotor, is the so-called 5' non translated region which is also involved in the initiation of transcription. In most cases the promotor will be located at the 5' end of the gene, but it can also vary in its position. The coding region is followed further downstream by the so-called 3' untranslated region. This region does contain signals which cause the termination of transcription and in eucargotic cells an additional signal that causes the polyadenylation of the transcribed RNA. The above constructed DNA sequence are examples for chimeric genes.
The DNA sequences which regulate the expression may be derived from different sources, e.g. plant, virus or bacterial genes which are active in plants. There are promotors which cause konstitutiv or inducible expression. Inducible promotors may be expressable depending on the development of the cell or tissue specific. Preferred promotors include the Cauliflower Mosaic Virus (CAMV) 35S promotor, the nopaline synthase promotor, the octopine synthase promotor and the ubiquitin promotor. The chimeric genes may also include leader sequences and signal sequences.
The DNA construct can be introduced into the plant cell using different techniques that are described in the art. These methods include direct gene transfer (EP 0 164 575), ballistic particle, microinjection and Agrobacterium mediated transformation (EP 0 116 718, Hoekema and An et al.).
The selection of plant cells which have been transformed is enabled by the use of a selectable marker gene which is also transferred. The expression of the marker gene confers a phenotypic trait that enables the selection. Examples for such genes are those coding for antibiotica or herbicide resistance, e.g. neomycin or phosphinothricin resistance.
Plants which can be protected may be either monocotyledons or dicotyledons.
Examples of families that are of special interest are Solanaceae and Brassicaceae. Examples of species of commercial interest that can be protected include:
tobacco, Nicotiana tabacum L. tomato, Lycopersicon esculentum Mill, potato, Solanum tuberosum L, petunia, Petunia hybrida (Solanaceae) Canola/Rapeseed, Brassica napus L, cabbage, broccoli, kale etc., Brassica oleracea L, mustards Brassica juncea L, - Brassica nigra L, and Sinapis alba L (Brassicaceae),
- sugar beet, Beta vulgaris, (Chenopodiaceae),
- cucumber, Curcurbita sp. (Curcurbitaceae),
- cotton, Gossypium sp., (Malvaceae),
- sunflower, Helianthus annuus,
- lettuce Lactuca sativa, (Asteraceae=Compositae),
- pea, Pisum sativum,
- soybean, Glycine max and alfalfa, Medicago sp. (Fabaceae = Leguminoseae),
- asparagus, Asparagus officinalis; - gladiolus, Gladiolus sp., (Lilaceae);
- corn, Zea mays and - rice, Oryza sativa (Poaceae).
In an preferred embodiment the gene prevents diseases of plants such as potato, tomato, wheat, cabbage and Chinese cabbage.
3. Examples
The invention is described in detail hereinafter, especially in its preferred embodiment by way of examples for non-limitive illustration purposes.
3.1 Legends to figures
Figure 1 : Protease activity of culture filtrates of Paecilomyces lilacinus (108 conidia) in 10 ml liquid medium (NM: minimal medium, CFM: corn flour medium) to which different substrates were added: E: 2,500,000 Meloidogyne hapla eggs for MME and 760,000 M. hapla eggs for CFME, N: nitrogen (NaN03 (0.2 %) + asparagine (0.2 %)) and NG: nitrogen (NaN03 (0.2 %) + asparagine (0.2 %)) + glucose (2.0 %). Figure 2: Protease activity / μg protein of culture filtrates of Paecilomyces lilacinus (mycelium after 6 days of growth in 250 ml MNNG) in 250 ml liquid medium (MN: minimal medium + nitrogen (NaN03 (0.2 %) + asparagine (0.2 %)) to which chitin was added as substrate. Glucose was added daily.
CHIT : colloidal chitin (0.2 % or 1.0 % (w/v),
GLUC : glucose (0.3 % (w/v) / day).
Figure 3: SDS-PAGE patterns of filtrates of 4 days old cultures of Paecilomyces lilacinus in minimal medium to which different substrates were added: Lane NG: Nitrogen + Glucose; Lane V: Vitellin; Lane C: Chitin; Lane E: Eggs; Lane M: Markers, Molecular weight of markers are indicated at the left side.
Figure 4: Protease activity of fractions of Bactracin-Sepharose column to which concentrated culture filtrate of Paecilomyces lilacinus, grown in minimal medium with chitin as substrate, was added. Elution was started at fraction 7. Procedure was described under Materials & Methods.
Figure 5: Protease activity of purified protease (1.875 μg) of Paecilomyces lilacinus at different pH. Measurements were performed in triplicate. Protease was purified as described under Materials & Methods from culture filtrate of P. lilacinus in minimal medium with vitellin as substrate.
Figure 6: Protease activity of purified protease (1.875 μg) of Paecilomyces lilacinus at different temperatures. Measurements were performed in triplicate. Protease was purified as described under Materials & Methods from culture filtrate of P. lilacinus in minimal medium with vitellin as substrate. Figure 7: Protease activity of purified protease (0.47 μg) of Paecilomyces lilacinus after incubation for 40 minutes at 4 °C with different inhibitors, SH-reducing agents and metal ions. Measurements were performed in triplicate. Final inhibitor concentration was 1 mM. Protease was purified as described under Materials & Methods from culture filtrate of P. lilacinus in minimal medium with vitellin as substrate. Protease- inhibitors were respectively none, PMSF, 1,10-phenanthroline, Pepstatine, E64, DTT, Cysteine, CaCI2, MgCI2 and EDTA.
Figure 8: Sequence of primer A and primer B. I: Inosine residue; Y: Cytidine or Thymidine; R: Adenine or Guanine.
Figure 9: Restriction map of pSP3, pSP4, pSP3-2. The probable location of the 240-bp PCR-generated fragment is shown. E, EcoRI; H, Hindi; H?, Hindi, location uncertain; N, Ncol; S, Smal; X, Xhol. No Avail, BamHI, Notl, Nrul, Pstl, Sad, Sacll sites, no internal EcoRI and Xhol sites.
Table 1 : Protease activity and protein content of filtrates of 4 days old culture of Paecilomyces lilacinus (2x108 conidia) in 200 ml liquid medium (MM: minimal medium) to which different substrates were added: MMC : colloidal chitin (0.2 % (w/v)),
MME : 2.250.000 Meloidogyne hapla eggs,
MMV : vitellin (0.2 % (w/v)),
MMNG: nitrogen (NaN03 (0.2 % (w/v)) + asparagine (0.2 %)
(w/v) + glucose (2.0 % (w/v). Culture filtrates were concentrated to 10 ml and dialyzed against demineralized water. Table 2: Protease activity and glucose-concentration of culture filtrate, and weighth of mycelium of 4 days old cultures of Paecilomyces lilacinus
(1.5x108 conidia) in 10 ml liquid medium (MM: minimal medium) to which different substrates were added:
MMC : colloidal chitin (0.2 % (w/v)),
MME : 450,000 Meloidogyne hapla, eggs,
MMN : nitrogen (NaN03 (0.2 % (w/v)) + asparagine (0.2 %
(w/v)), MMG : glucose (2.0 % (w/v)).
Culture filtrate was supplemented to 15 ml with sterile demineralized water.
3.2. Material & Methods 3.2.1 Culture of fungus
Paecilomyces lilacinus (Thorn.) Samson (CBS 143.75), obtained from the CBS (Central Bureau of Fungal Cultures) in Baarn (The Netherlands), was routinely maintained (once a month) on Potato Dextrose Agar (PDA; difco laboratories) in 90 mm petridishes at 25 °C. A conidial suspension was obtained by adding 5 ml of sterilized water to a PDA petridish containing sporulating mycelium and scraping the surface with a glass rod. Liquid cultures were obtained by inoculating conidia of the fungus to minimal salt medium or corn flour medium supplemented with the substrate. The minimal salt medium (MM) consisted of 4.56 gr I^HPO^ 2.77 gr KH2HP04, 0.5 gr MgS04. 7H20 and 0.5 gr KCI / liter pH 6.0.
The corn flour medium (CFM) was prepared by steaping 40 gr of grinded corn flour in 1 I demineralized water for 1 hour at 55 °C. Next the mixture was filtered over Whatman no 1 filter and the filtrate was used after autoclavation (20 minutes 120 °C). The following substrates were added alone or in combination when required: vitellin (0.2 % (w/v); Sigma), collodial chitin (0.2 % and 1.0 % (w/v); prepared according to Lingappa & Lockwood (1962) using chitin from Sigma), root-knot nematode eggs ( > 400.000; isolated as described below), nitrogen (N) (as 2.0 gr asparagine + 2.0 gr NaN03/l) and glucose (G) (2.0 % (w/v).
In one experiment mycelium of Paecilomyces lilacinus was used as inoculum. Mycelium was obtained by centrifuging a 6 day old culture of conidia of P. lilacinus in MMNG for 45 minutes at 9000 g. Cultures were grown in a shaking waterbath for several days at 30 °C and 125 strokes per minute. Culture filtrates were obtained by centrifuging cultures for 45 min at 9000 g. Supernatants were concentrated using an Amicon (YM-10 filter) and clarified through a 0.22 μm-filter (Schleicher & Schuell). Pellets were freeze dried and the weigth of the mycelium was measured.
To obtain culture filtrates for daily testing of protease activity, protein content and glucose concentration 1 ml aliquots were removed from the culture and centrifuged for 15 minutes at 14.000 g. The resulting supernatant was clarified through a 0.22 μm-filter (Schleicher & Schuell).
Escherichia coli cells were grown in LB broth (1 % bacto tryptone, 0.5 % yeast extract, 0.5 % Sodium chloride), when necessary 100 μg/ml ampicillin was added. E. coli strain PLK-F'(mrcA-, mrcB-, recA-, TetR) was used for the preparation of plating cells. E. coli strain InVaF' competent cells were purchased from Invitrogen (San Diego) and used as carrier of recombinant plasmids.
A spore suspension of Paecilomyces lilacinus was obtained by adding 5 ml of sterilized water to a PDA plate containing sporulating P. lilacinus mycelium and scraping the surface with a glass rod. Liquid cultures for genomic DNA isolations were grown in potato dextrose broth (PDB; Difco). The liquid induction medium (IM) used for the isolation of the mRNA fraction contained 0.5 gr/L KCI, 0.5 gr/L MgS04, 1.36 gr/L KH2P04, 2.28 gr/L K2HP04 and 4x108 nematode eggs/L as the sole carbon and nitrogen source. As an inoculum 4x1010 spores/L was used. 3.2.2 Culture of nematode
The root-knot nematode Meloidogyne hapla Chitwood, obtained from the Centre for Plant Breeding and Reprodcution Research (CPRO-DLO) in Wageningen, was continiously maintained in a greenhouse on tomato plants (Lyopersicon esculentum cv. Moneymaker) for 8 hr at 15 °C and 16 hr at 20 °C per day with a relative humidity of > 65%. Artificial lighting was supplied to give 16 hours daylength. The plants are grown in sterilised silver sand and nutrients were supplied according to Steiner.
The Northern root-knot Meloidogyne hapla was reared on tomato plants (Lycopersicon esculentum cv. Moneymaker) in a greenhouse. Six week-old plantlets were inoculated by placing 2000 nematode eggs in the vicinity of the stem. Eight to ten weeks after inoculation, nematode eggs were collected by vigorously shaking the nematode-infected roots in 1 % (v/w) hypochlorite for 10 min. The liberated eggs were collected by centrifugation (2x103 rpm, 10 min), and loaded onto a continous sucrose gradient, prepared by freezing and slowly thawing a 40 % (w/v) sucrose solution. The gradient was spun (2x103 rpm, 10 min) and the eggs were collected, washed several times with sterilized, distilled water, and counted.
3.2.3 Isolation of nematode-eggs.
Eggs of the root-knot nematode M. hapla were isolated according to Schaad & Walker (1975). Roots of tomato plants infected with the root-knot nematode M. hapla for more than 6 weeks were rinsed with water to remove the soil. Then they cut into small parts and shaken with 1.0 % hypochlorite for 5-10 minutes. Eggs were collected from the solution by sequential passage over 200,75 and 22 μ sieves (Retsch (200 and 75 μ) and Scrynel (22 μ) and separated plant material through sucrose gradient centrifugation (0-30 % sucrose and 5 minutes centrifugation at 1600 g in sterile 50 ml Greiner tubes). Ultimately eggs (on top of the gradient) were washed several times with sterile water. 3.4 Protease activity
Protease activity was determined by a modified procedure of Rinderknecht et al. (1968). Ten mg of Hide Powder Azure (Sigma) was incubated in 50 mM Sodium acetate buffer pH 6.0 with the sample in a final volume of 3 ml in small glass containers. Incubation was at 37 °C in a shaking waterbath (110 strokes/min) till the solution turned blue (between 5 and 30 minutes). Following incubation the samples were put on ice and centrifuged in 1.5 ml Eppendorf cups for 5 minutes at 14.000 g. The absorbance of the supernatant was measured at 595 nm on a Uvikon 940 spectrophotometer. Protease activity of the sample was measured in duplo, averaged and expressed as A5g5 ml*1hr"1 after correction for the blank (Milli Q water).
Protease activity on gelatine agar plates was determined by incubating conidia of P. lilacinus on 1.2 % (w/v) NM agar at 25 °C containing 0.2 % (w/v) gelatin. Halo's indicating extracellular proteolytic activity were visualized by staining with Coomassie Brilliant Blue.
Protein quantification of the samples were determined according the Bradford (1976), using BSA (bovine serum albumine) as the standard.
Concentration of D-glucose of the samples were determined with the enzymatic UV-method of Boehringer (Cat.no. 139106).
SDS-PAGE was performed by the method of Laemmli (1970). Culture filtrate samples were precipitated with trichloroacetic acid, washed with ice-cold aceton, dried, suspended in sample-buffer (62.5 mM Tris/HCI pH 6.8, 10 % glycerol, 2 % SDS, 10 mM DTT, 0.1 % BPB) and boiled for 5 minutes. Native PAGE in the presence of gelatin was performed according to Heussen & Dowdle (1980). The concentrated culture filtrate was dialyzed overnight at 4 °C against equilibration buffer (100 mM Ammoniumacetate, 10 mM CaCI2, pH 6.5) and applied to an equilibrated 2 ml Bacitracin-Sepharose column. The Bacitracin-Sepharose was made as described by Stepanov & Rudenskaya (1983). After washing the column with 100 mM Ammoniumacetate buffer pH 6.5, the bound protease was eluted with elution buffer (100 mM Ammoniumacetate, 1 M NaCI, 25 % isopropanol pH 6.5). Fractions of 1 ml were collected and tested for proteolytic activity. Positive fractions were pooled and dialyzed agianst 100 nM Ammonium acetate pH 6.5.
3.2.5 Characterization of the protease
- Molecular weigth: After SDS-PAGE gels were stained with silver (Morrissey (1981)) and dried with the Kem-en Tec gel drying frame. Densitograms of the gels were obtained with the video densitometer Model 620 of BioRad. Molecular weight was determined using BioRad's Low Molecular Weight standards as reference (rabbit muscle phosphorylase b (97,400 Da), bovine serum albumine (66,200 Da), hen egg white ovalbumine (45,000 Da), bovine carbonic anhydrase (31,000 Da), soybean trypsin inhibitor (21 ,500 Da) and hen egg white lysozyme (14,400 Da)).
- Optimum pH: For the determination of the optimal pH for protease activity the following buffers were used: pH 4.0 - 5.7 Sodium acetate pH 6.0 - 7.6 Potassium phosphate pH 7.7 - 9.0 Tris/HCI pH 9.2 - 10.6 2-Amino-2-methyl-1-propanol/HCI pH 10.6 - 12.6 2-Amino-2-methyl-1-propanol/HCI
Protease activity was performed as described above in triplicate.
- Optimum temperature: For the determination of the optimum temperature for protease activity the proteolytic activity measurements were performed as described above in triplicate at different temperatures. - Isoelectric point: For the determination of the isoelectric point the protease was applied to a Mono P chromatofocusing column (HR 5/5 from Pharmacia) at pH 10.2 (25 mM 2-amino-2-methyl-1-propanol/HCI). With an FPLC (Fast Protein Liquid Chromatography) system gradient elution was performed with Polybuffer (1 :10) pH 8.0.
Iso-electrofocusing (IEF) was employed with the Phastsystem of Pharmacia using lEF-gels with a pH-gradient from 3 to 10 according to the instructions of the manufactory.
- Protease inhibition: Several protease inhibitors, SH reducing agents and metal ions were tested on the proteolytic activity of the protease: PMSF, E64, 1.10- phenantroline, pepstatine (Sigma); EDTA, cystein MgCI2 and CaCI2 (Merck); DTT (BioRad).
The purified enzyme was incubated for 40 minutes at 4 °C with inhibitor. After incubation the protease-activity was determined as previously described. Final inhibitor concentration was 1 mM. Protease activity in the absence of inhibitor was expressed as 100 %.
- Binding of protease to root knot nematode eggs: Root-knot nematode eggs were incubated at room temperature with the purified protease in 1.5 ml Eppendorf cups in 100 mM Potassium phosphate buffer pH 7.0 with continously shaking.
On several time points 200 μl samples were taken, centrifuged for 1 minute at 14.000 g. Protease activity and SDS-PAGE was performed on the supernatant. The bound enzyme was eluted from the nematode eggs with 100 mM Potassium phospate buffer containing 0.5 M NaCI.
- Degradation of vitellin by the serine protease: Vitellin (1 mg) was incubated with the purified protease for 16 hours at 37 °C in 1.5 ml Eppendorf cups in 100 mM Sodiumacetate buffer pH 6.0 (final volume was 1 ml). After incubation the samples were centrifuged (1 minute 14000 g) and absorption of the supernatant was mesaured at 280 nm on an Uvikon 940 spectrophotometer.
3.2.6 Recombinant DNA techniques.
All restriction enzymes and T4 ligase were obtained from Gibco BRL and used as recommended by the supplier. Methods were performed as described in Maniatis et al. (1982).
3.2.7 Isolation of genomic DNA from P. lilacinus
P. lilacinus was grown for 2-4 days in PDB at 30 °C and 150 rpm. The mycelium was collected by filtration through myracloth (Calbiochem Corporation, La Jolla). The mycelium was frozen in liquid nitrogen and grinded to a fine powder in a mortar. Per gram powder, 5 ml of extraction buffer (0.1 M NaCI, 10 mM Tris-HCI (pH 7.5), 1 mM EDTA, 1 % SDS) was added and the suspension was vortexted for 5 min. An equal volume of phenol/chloroform (1 :1) was added and the phases were vigourously mixed.
After centrifugation (20 min, 104 rpm), 0.1 volumes of 3 M Natriumacetat (NaAc, pH 5.2) and 2 volumes of ethanol were added to the aqueous phase. After precipitation (30 min, -20 °C), the nucleic acid fraction was pelleted by centrifugation (20 min, 104 rpm). The pellet was resuspended in 2 ml of water.
RNAse was added to a final concentration of 10 μg/ml and the solution was incubated at 37 °C for 30 min. The solution was extracted once with phenol/chloroform (1 :1), once with chloroform and then precipitated with ethanol. 3.2.8 Isolation of mRNA from P. lilacinus
P. lilacinus was grown for 48 hours in MM at 30 °C and 150 rpm. The mycelium was collected by filtration through myracloth. The mycelium was frozen in liquid nitrogen and grinded to a fine powder in a mortar. Per gram powder, 5 ml of extraction buffer (0.1 M NaCI, 10 mM Tris-HCI (pH 7.5), 1 mM EDTA, 1 % SDS) was added and the suspension was vortexted for 5 min. An equal volume of phenol/chloroform (1:1) was added and the phases were vigourously mixed. After centrifugation (20 min, 104 rpm), 0.1 volumes of 3 M NaAc (pH 5.2) and 2 volumes of ethanol were added to the aqueous phase. After precipitation (30 min, -20 °C), the nucleic acid fraction was pelleted by centrifugation (20 min, 104 rpm). The pellet was resuspended in sterile, distilled water and the mRNA fraction was isolated, using the polyATract system (Promega, Madison). Approximately 15 μg of mRNA was obtained from 200 ml culture. Using the lambda ZAP system (Stratagene, La Jolla) a cDNA library was contructed starting with 5 μg mRNA. The library was found to contain 500.000 individual clones.
3.2.9 Polymerase chain reaction
The PCR reaction mixture of 100 μL contained 50 mM KCI, 10 mM Tric/HCI (pH 8.3), 0.5-2 mM MgCI2, 100 μM of each dNTP, 100 pmol of oligonucleotide A and oligonucleotide B (see below), 400 ng of genomic DNA from P. lilacinus, and 2.5 units of AmpliTaq DNA polymerase (Perkin Elmer) mineral oil was added to prevent evaporation. Each of the 35 amplification cycles included a denaturation step at 94 °C for 1 min, an annealing step at 42 °C to 60 °C for 2 min, and a chain elongation step at 72 °C for 3 min. The amplification reaction was preceded by a denaturation step at 94 ° for 0.5 min, and the elongation step of the last cycle was extended to 5 min. 3.2.10 Screening of the cDNA library
The library was plated at a density of 10.000 plaques/plate on PLK-F' cells. After overnight incubation at 37 °C, duplicate nitrocellulose filters or each plate were prepared according to Maniatis et al (15).
The filters were baked at 80 °C for 2 hours and incubated in prehybridisation solution (6x SSC, 5x Denhardt's solution, 0.1 % SDS, 100 μg/mL denatured salmon sperm DNA) at 65 °C for 4 hours.
To obtain a radioactive probe, 1 μL of the PCR reaction product was used. The labelling reaction mixture was identical to the PCR reaction mixture, except that the dATP was substituted for 5 μL alpha-32P-dATP (3000 Ci/mmol, 10 mCi/ml). To incorporate the radioactive nucleotides, 7 amplification cycles were performed identical to the cycling conditions used to obtain the DNA fragment. The probe was separated from the free nucleotides using a Sephahdex G-50 spin column. After denaturation (10 min, 100 °C), the probe was added to prehybridisation solution and the filters were hybridized for 16 hours. The filters were washed twice 2x SSC/0.1 SDS for 30 min at 65 °C, and once with 0.2x SSC/0.1 SDS for 30 min at 65 °C. X-ray film (Fuji RX) was exposed to the filters for 16 hours and an autoradiograph was obtained. Positives were rescreened by the same procedure until pure. After in vivo excision of the plasmids according to the manual supplied by the manufacturer (Stratagene), they were subjected to restriction enzyme analysis.
3.2.11 Sequence analysis
The insert of pSP3 was partially sequenced using the Taq dye primer cycle sequencing kit (Applied Biosystems, Foster City) and an Biorad R370 automated sequencer, and the T7 sequencing kit and the Automated Laser Fluorescent DNA Sequencer of Pharmacia. 3.3 Working Examples
The nematode egg-parasitic fungus Paecilomyces lilacinus penetrates the egg-shell of the root-knot nematode Meloidogyne hapla which consists for a great part of proteins. In the following induction, purification and characterization of this protease is described.
3.3.1 Induction of the protease
Paecilomyces lilacinus grown for 3 days at 25 °C on solid agar containing gelatin showed halo's after staining with Coomassie Brilliant Blue proving extracellular production of proteases. Subsequently the production of extracellular protease by the fungus in liquid minimal salt medium (MM) and in liquid corn flour medium (CFM), to which several substrates were added, was studied. Protease activity of the culture filtrate was monitored at daily intervals following inoculation of the medium with conidia of the fungus. Figure 1 shows the protease activities of the culture filtrate with eggs (E) as substrate and with nitrogen and glucose (NG) as control on several days after inoculation. Even after two days protease activity was increased in CFM cultures containing eggs (CFME). It reaches maximum at 3 days after inoculation and decreases to zero protease activity on day 7. On day 3 MM cultures containing eggs (MME) showed also a substantial increase in proteolytic activity. On day 10 activity was still increasing. MM supplemented with nitrogen and glucose (MNNG) showed an increase of protease activity similar to MME. Addition of chitin (C) or vitellin (V) as substrate to cultures of P. lilacinus in MM resulted also in induction of proteolytic activity. Table 1 shows protease activity and protein content of the filtrate of 4 different cultures of P. lilacinus after 4 days growth. The highest induction of protease activity was seen with vitellin as substrate. Specific protease activity (protease activity/ μg protein) was highest in cultures containing eggs as substrate. To see whether the fungus used glucose as carbon sourece, glucose concentrations were determined in the culture filtrate, of the fungus in MNNG (Figure 1).
After day 4 almost all glucose was used. Glucose is a well known repressor of the induction of many enzymes. To study if catabolite repressioh was involved in the induction of P. lilacinus protease, glucose was added at a concentration of 0.3 % each day to MNNC. In this experiment mycelium of P. lilacinus was used as inoculum.
Figure 2 shows that the induction of protease activity was repressed by glucose. Adding conidia or mycelium (harvested after incubating the same amount of conidia of P. lilacinus as inoculum in MNNG for 6 days) resulted in no differences in induction of the proteolytic activity.
To determine the influence of nitrogen in the medium on the induction of protease nitrogen was added to MMGC or to MMGE. The results after 4 days of growth are shown in Table 2. When there is no nitrogen and no substrate present in the medium (MMG) no proteolytic activity could be detected in the culture filtrate and no glucose had been consumed. There was no growth of the fungus. In the presence of substrate (C, E) protease activity was observed and glucose had been used. In the culture with chitin as substrate (MMGC) glucose concentration decreased to 50 %, with eggs as substrate (MMGE) it decreased to 90 %. When nitrogen was present in the medium and no substrate (MNNG), glucose concentration lowered to 15 % with low protease activity. Addition of eggs as substrate (MMNGE) resulted in high protease activity and complete glucose consumption. Chitin as substrate (MMNGC) resulted in low protease activity and also complete glucose comsumption. The weigth of mycelium was highest in MMNGC. Specific protease activity (protease activity per mg mycelium) was highest in MMGE. Table 1 Protease activity - Protein content
Medium Protease activity Protein content Specific Protease Activity
(A595 l'1 1 hr"1) μg ml'1) (A595 ml"1 hr"1)
MMNG 4.24 47.5 0.089
MMC 23.85 104.2 0.229
MME 21.83 35.3 0.618
MMV 93.79 303.9 0.309
Table 2 Protease activity - Glucose concentration
Medium Protease Glucose Weight Specific
Activity Cone. Protease Activity
(A595 ml"1 hf1) (%) (mg) (A595 ml"1 hr"1 m9 mycelium"1) x 1000
MMG 0.06 2.12 20 2.79
MMGE 5.79 1.80 50 115.76
MMGC 5.64 1.04 100 56.36
MMNG 1.37 0.29 180 7.59
MMNGE 5.38 0.006 200 26.91
MMNGC 1.46 0.002 350 4.17
3.3.2 Purification of the protease
The SDS-PAGE patterns of the culture filtrates showed many proteins produced in the different cultures (Figure 3). The protein pattern of the culture filtrate with vitellin as substrate looked very similar to the one of the culture filtrate with nematode eggs as substrate (lane 3 and 5 respectively). To identify the nature of the protease the proteolytic activity in the culture filtrate was inhibited with several protease inhibitors. Inhibition of the proteolytic activity in the culture filtrate with PMSF (a serine protease inhibitor) showed that most of the protease activity present in the medium was inhibited suggesting a serine protease being involved as the most secreted protease. Purification of the serine protease(s) was first performed with Benzamidine-Sepharose, an affinity resin for serine proteases of class I (Pharmacia). Since all of the proteolytic activity did not bind, the protease belonged to another serine protease class, the subtilisin class. Stepanov & Rudenskaya (1983) showed that this class of proteases binds to Bacitracin-Sepharose.
Figure 4 shows the proteolytic activity of fractions of the Bacitracin-Sepharose column to which concentrated culture filtrate of the fungus (grown for 4 days in MM with chitin a substrate) was applied. After washing and elution the protease activity was predominantly found in fractions 8 and 9. Integration of the densitogram of fraction 8 revealed that one protein was present for more than 85 %. It was concluded that this was the serine protease. The same protein was purified from culture filtrates of P. lilacinus grown with eggs and vittelin as substrate. All substrates induced the serine protease.
3.3.3 Characterization of protease
The molecular weight of the serine protease of Paecilomyces lilacinus using molecular weigth markers was 33.5 kDa. Since the protease did not bind to the Mono P column at pH 10.2 the isoelectric point should even be higher. Isoelectric focussing using the Phastsystem of Pharmacia and pH 3-10 gels showed that the protease focussed at pH 10.
The optimum pH and temperature for proteolytic activity was obtained by performing protease activity measurements at different pH and temperature respectively. Figure 5 shows an optimum pH of 10.3 after fitting of the protease activity curve. Figure 6 shows the temperature activity profile for the P. lilacinus protease. Optimum temperature for the proteolytic activity was about 60 °C.
In order to elucidate the type of protease involved a series of inhibitors, SH-reducing agents and some metal ions were tested on the proteolytic activity of the protease. Figure 7 shows the effect of several protease inhibitors, some SH- reducing agents and some metal ions on the activity of the purified protease. PMSF inhibited the activity for 100 %. Consequently the protease is of the serine protease class. All other protease inhibitors tested did not effect the proteolytic activity of the enzyme significantly. DTT and cystein did not influence its activity also suggesting no SH-groups being involved in the active center of the enzyme. Ca+ +- and Mg+ +- ions lowered the protease activity slightly. EDTA on the other hand enhanced its activity suggesting a negative influence of metal ions present in the assay.
The purified protease was capable of degrading insoluble vitellin and produced halo's on MM agar plates containing gelatin.
Incubation of the protease with nematode eggs at room temperature showed that the protease bound quantitatively to the eggs shown by activity measurements on the supernatant and that the eggs floated after overnight incubation. Control eggs did not. The bound protease could be eluted from the eggs by 0.5 M CaCI as shown by activity measurements and SDS-PAGE. 3.3.4 Design of oligonucleotides
The method used for the isolation of cDNA clones encoding the P. lilacinus serine protease (Psp) was based on the amplification of a part of the gene by the polymerase chain reaction. The design of the set of oligonucleotides used in the PCR reactions, was based on both amino acid sequence data on the Psp protein and on amino acid sequences which are conserved among subtilisin-like proteases.
The 16 amino acids of the N-terminus of Psp were determined to be Ala-Tyr-Thr- Gln-Gln-Pro-Gly-Ala-Pro-(His?/Cys?/Trp?)-Gly-Leu-Gly-Arg-lle-(Ser). Comparison of this sequence to other amino acid sequences of fungal subtilisin proteases, as compiled by Tatsumi et al., showed that the C-terminal part of the 15-amino acid stretch contained a conserved region, whereas the N-terminal part showed little homology.
The first oligonucleotide was based on this stretch of 9 amino acids to avoid cross- reactions with other subtilisin-like proteases that P. lilacinus may produce (figure 8).
Panel A. Panel B.
A GLT TQK SAP WGL SIS DSI GHG THV SGT
B EFD TQN SAP WGI ARIS DGN GHG THC AGT
C AIQ TTP VTQ WGL SRIS DLL GHT HVA GT
D AAQ TNA PWG LAR IS DGN GHG THC AGT
E AYT QQP GAP WGL GRIS DGN GHG THC AGT
Panel A: Comparison of the N-terminal sequence of Psp to the N-termini of other subtilisin-like protease. A, Aspergillus oryzae Alp (Tatsumi et al.); B, Saccharomyces cerevisiae protease B (Moehle et al.); C, Yarrowia lipolytica alkaline protease (Davidow); D, Tritirachium album, proteinase K (Jany et al.); E, Paecilomyces lilacinus Psp. Panel B: Comparison of the amino acid sequences surrounding the active site His residue (see also figure 8: indicated by *). A-D, as in panel A; E, hypthetical sequence used for the design of oligonucleotide B.
To avoid strong degeneration of the oligonucleotides, inosine residues were used where four-base wobble occured. Degeneracy was allowed at sites where two-base wobble occured.
Primer A: 5' GCITAYACICARCARCCIGGIGCICC 3' Primer B: 5' GTICCIGCRCARRGIGTICCRTGICCRTTICC 3*
Sequence of primer A and primer B. I, inosine residue; Y, C or T; R, A or G (see also Figure 8).
Matrix comparison of amino acid sequences of proteases of the subtilisin family reveals several highly conserved regions, including the regions surrounding the three residues (Asp 32, His 64 and Ser 221 in subtilisin BPN, Markland and Smith (1967)) composing the catalytic triad. The distance between the N-terminus and the active site His-residue is 69-77 amino acids in fungal subtilisins.
Subsequently a comparison of the complete amino acid sequence (see also sequence protocol, SEQ ID NO 2) with several mature subtilisin-like serine protease confirmed that three amino acids (aspartic acid, histidine and serine) are involved in the active center of the serine protease.
The complete mature protein sequence was aligned using the GCG Sequence Analysis Software Package. The sequence was compared with a thermo-stable serine protease of Tritirachium album Limber (Samal et al. (1990)); an alkaline protease of Aspergillus fumigeius (Jaton-Ogay et al. (1992)); an basic proteinase of Trichoderma harzianum (Geremia et al. (1993)); an alkaline protease of Acremonium chysogenium (Isogai et al. (1991)); aqualysin I of Thermus aquaticus (Terada et al. (1990)); protease B of Saccharomyces cerevisiae (Moehle et al. (19987)) and an alkaline extracellular protease of Yarrowia lipolytica (Davidow et al. (1987)).
3.3.5 Polymerase chain reactions
To obtain a single reaction product, the temperature of the annealing step of the cycles was varied from 42 °C to 60 °C, resulting in multiple bands in all experiments. The annealing temperature was set at 60 °C and the MgCI2- concentration was optimized. Using a MgCI2-concentration of 0.5 mM, a single DNA band was seen on an agarose gel. The size of the band was estimated to be 240 basepairs. Although several other bands were seen at higher MgCI2- concentrations, the 240-bp band was always the most predominant.
This is approximately the expected size, assuming that the distance between N- terminus and the active site histidine residue in Psp is similar to that of other fungal subtilisin-like proteases (69-77 amino acid residues).
3.3.6 Screening of cDNA library.
The PCR reaction product containing the single visible 240-bp DNA band was used a radioactive labelled probe and hybridised to duplicate nitrocellulose filters containing 100.000 plaques in total. The resulting autoradiographs showed both strong and weak hybridisation signals.
Seven strongly hybridizing plaques were identified and subsequently purified. Using the in vivo excision system offered by the lambda ZAP system, the 7 lambda clones were converted to plasmids. 3.3.7 Restriction enzyme analysis
A restriction map was made of the 7 plasmids, and based on these data they could be classified in two categories. The first category, consisting of 4 plasmids, contained an insert of 1400 base pairs. One of these plasmids, which was used in further studies, was named pSP3. The second category, consisting of 3 plasmids, contained an insert of 1200 bp. One of these plasmids was named pSP4. Based on restriction patterns, we concluded that the insert of pSP4 is a shorter version of pSP3 (figure 9).
To facilitate sequencing of the complete insert of pSP3, a subclone, pSP3-2, was created by digesting pSP3 with Smal and religate the larger fragment containing the vector and 0.5 kb of the insert. +
3.3.8 Sequencing
pSP3 was partially sequenced. It was possible to identify the region containing oligonucleotide A (figure 9). This region is located 250 bp downstream of the EcoRI cloning site. Furthermore, a 3'poly(A) sequence was found.
The region just downstream of oligonucleotide A is reasonably consistent with the known N-terminal amino acid sequence of Psp, which leads us to believe that pSP3 encodes the Psp protease of P. lilacinus. The length of the insert of pSP3 upstream of the oligonucleotide A sequence suggests that this is a full length or nearly full length cDNA.
The whole mature protein sequence is included (see SEQ ID NO 1 and 2, the protein sequence starts at amino acid No. 84 with Ala-Tyr-Thr, see also 3.3.4). The start codon and part of the leader sequence are therefore missing. 4. References.
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RINDERKNECHT, H., GEOKAS, M.C., SILVERMAN, P. & HAVERBACK, B.J. (1968). A new ultrasensitive method for the determination of proteolytic activity. Clinica Acta 21 , 197-203.
SAMAL, B.B., KARAN, B., BOONE, T.C., OSSLUND, T.D., CHEN, K.K. & STABINSKY, Y. (1990). Isolation and characterization of the gene encoding a novel thermostable serine proteinase from the mould Tritirachium album Limber. Molecular Microbiology 4, 1789-1792.
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ST. LEGER, R.J., COOPER, R.M. & CHARNLEY, A.K. (1986a). Cuticle-degrading enzymes of entomopathogenic fungi: Cuticle degradation in vitro by enzymes from entomopathogens. Journal of Invertebrate Pathology 47, 167-177.
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(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Research Institute for Plant Protection
(IPO-DLO)
(B) STREET: Binnenhaven 12 / Postbus 9060 (C)' CITY: Wageningen
(D) STATE: -
(E) COUNTRY: Netherland
(F) POSTAL CODE (ZIP): 6700 GW
(G) TELEPHONE: 08370-76000 (H) TELEFAX: 08370-10113 (I) TELEX: 45888 intas nl
(ii) TITLE OF INVENTION: New alkaline serine protease of Peacilomyces lilacinus
(iii) NUMBER OF SEQUENCES: 14
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1332 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Paecilomyces lilacinus
(ix) FEATURE:
(A) NAME/KEY: intron
(B) LOCATION: 1..1332
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GGCACGAGCT CCTCTCCTGA CGCCCCGCGG CGCCAGCAGC TCATCAACGG CAAGTACATT 60
GTCAAGTTCA AGGACGGCAT GTCCATCGCC TCTGTCGACA AGACTGTCAG CGCTCTGTCC 120
TCGAAGGCCG ACCGCGTCTA CAACCACATT TTCCGAGGCT TCGCGCAACC TGAATGCCAA 180
CGACCTCAAG ACCCTGCGCG ACCACCCTGA TGTCGAGTAC ATTGAGCAGG ATGCCATAAT 240
CACCATCAAC GCGTACACTC AGCAGCCCGG CGCCCCCTGG GGTCTCGGAC GCATCTCTCA 300
CCGCAGCAAG GGTAGCACCA CATACGAGTA TGATACCAGC GGCGGCAGTG GCACCTGCGC 360
CTATGTCATC GACACTGGCG TCGAGGCTTC GCACCCCGAG TTCGAGGGCC GCGCCAGCCA 420
GATCAAGAGC TTCATCAGCG GCCAGAACAC CGACGGCAAC GGCCATGGCA CTCACTGCGC 480
CGGCACCATC GGCTCCAAGA CGTACGGTGT TGCCAAGAAG ACCAAGATCT ACGGTGTCAA 540
GGTCCTCGAC AACTCGGGCT CCGGCTCATA CTCGGGCATC ATCTCCGGTA TGGACTTTGC 600 CGTTCAGGAC TCCAAGTCGC GCAGCTGCCC CAAGGGTGTC GTCGCCAATA TGTCTCTGGG 660
CGGTGGAAAG GCTCAGTCCG TCAACGACGG TGCCGCTGCC ATGATCAGGG CCGGCGTCTT 720
CCTCGCCGTC GCCGCTGGCA ACGACAACGC TAACGCCGCC AACTACTCCC CTGCCTCTGA 780
GCCGACTGTT TGCACCGTCG GCGCCACCAC CTCTTCTGAT GCGCGATCTT CGTTCTCCAA 840
CTACGGCAAT CTCGTCGACA TCTTCGCCCC GGGTAGCAAC ATTCTGTCCA CCTGGATCGG 900
TGGCACTACC AACACCATCT CTGGTACTTC CATGGCCACT CCCCACATTG TTGGTCTCGG 960
CGCCTACCTC GCCGGTCTGG AGGGTTTCCC CGGCGCCCAG GCGCTCTGCA AGCGCATCCA 1020
GACCCTTTCT ACTAAGAACG TCCTCACCGG CATCCCCAGC GGCACTGTCA ACTACCTCGC 1080
CTTCAACGGC AACCCCAGCG GCTAAATGCT ACAAGAATGA GGACTCCTCG ACGACCTTTA 1140
TGCCAGCCAT GACCTTTTTT GGAGCATATC AACAGCGGCG AGATCCTCTT GGGACAAAGG 1200
TAAGTATGTG GCATTGAACT GCGCTCCTGT CATATACTCC GCGCAAGGAA GAGTAAAAAG 1260
TGGACATGTC TTTTAAATAG CACCAATACA TATCTTATGA GGCCTCGAAA AAAAAAAAAA 1320
AAAAAAAAAA AA 1332 (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 367 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Paecilomyces lilacinus
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 1..367
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Ala Arg Ala Pro Leu Leu Thr Pro Arg Gly Ala Ser Ser Ser Ser Thr 1 5 10 15
Ala Ser Thr Leu Ser Ser Ser Arg Thr Ala Cys Pro Ser Pro Leu Ser 20 25 30
Thr Arg Leu Ser Ala Leu Cys Pro Arg Arg Pro Thr Ala Ser Thr Thr 35 40 45
Thr Phe Ser Glu Ala Ser Arg Asn Leu Asn Ala Asn Asp Leu Lys Thr 50 55 60
Leu Arg Asp His Pro Asp Val Glu Tyr lie Glu Gin Asp Ala lie lie 65 70 75 80
Thr lie Asn Ala Tyr Thr Gin Gin Pro Gly Ala Pro Trp Gly Leu Gly 85 90 95
Arg lie Ser His Arg Ser Lys Gly Ser Thr Thr Tyr Glu Tyr Asp Thr 100 105 110
Ser Gly Gly Ser Gly Thr Cys Ala Tyr Val lie Asp Thr Gly Val Glu 115 120 125 Ala Ser His Pro Glu Phe Glu Gly Arg Ala Ser Gin He Lys Ser Phe 130 135 140
He Ser Gly Gin Asn Thr Asp Gly Asn Gly His Gly Thr His Cys Ala 145 150 155 160
Gly Thr He Gly Ser Lys Thr Tyr Gly Val Ala Lys Lys Thr Lys He 165 170 175
Tyr Gly Val Lys Val Leu Asp Asn Ser Gly Ser Gly Ser Tyr Ser Gly 180 185 190
He He Ser Gly Met Asp Phe Ala Val Gin Asp Ser Lys Ser Arg Ser 195 200 205
Cys Pro Lys Gly Val Val Ala Asn Met Ser Leu Gly Gly Gly Lys Ala 210 215 220
Gin Ser Val Asn Asp Gly Ala Ala Ala Met He Arg Ala Gly Val Phe 225 230 235 240
Leu Ala Val Ala Ala Gly Asn Asp Asn Ala Asn Ala Ala Asn Tyr Ser 245 250 255
Pro Ala Ser Glu Pro Thr Val Cys Thr Val Gly Ala Thr Thr Ser Ser 260 265 270
Asp Ala Arg Ser Ser Phe Ser Asn Tyr Gly Asn Leu Val Asp He Phe 275 280 285
Ala Pro Gly Ser Asn He Leu Ser Thr Trp He Gly Gly Thr Thr Asn 290 295 300
Thr He Ser Gly Thr Ser Met Ala Thr Pro His He Val Gly Leu Gly 305 310 315 320
Ala Tyr Leu Ala Gly Leu Glu Gly Phe Pro Gly Ala Gin Ala Leu Cys 325 330 335
Lys Arg He Gin Thr Leu Ser Thr Lys Asn Val Leu Thr Gly He Pro 340 345 350
Ser Gly Thr Val Asn Tyr Leu Ala Phe Asn Gly Asn Pro Ser Gly 355 360 365
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Aspergillus oryzae
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 1..15
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Gly Leu Thr Thr Gin Lys Ser Ala Pro Trp Gly Leu Ser He Ser 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Saccharomyces cerevisiae
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 1..16
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Glu Phe Asp Thr Gin Asn Ser Ala Pro Trp Gly He Ala Arg He Ser 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Yarrowia lipolytica
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 1..16
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Ala He Gin Thr Thr Pro Val Thr Gin Trp Gly Leu Ser Arg He Ser 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Tritirachium album
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 1..14
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Ala Ala Gin Thr Asn Ala Pro Trp Gly Leu Ala Arg He Ser 1 5 10
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Paecilomyces lilacinus
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 1..16
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Ala Tyr Thr Gin Gin Pro Gly Ala Pro Trp Gly Leu Gly Arg He Ser 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Aspergillus oryzae
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 1..12
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Asp Ser He Gly His Gly Thr His Val Ser Gly Thr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Saccharomyces cerevisiae
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 1..12
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Asp Gly Asn Gly His Gly Thr His Cys Ala Gly Thr 1 5 10
(2) INFORMATION FOR SEQ ID NO: 10:
(i) "SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Yarrowia lipolytica
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 1..11
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Asp Leu Leu Gly His Thr His Val Ala Gly Thr 1 5 10
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Tritirachiu album
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 1..12
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
Asp Gly Asn Gly His Gly Thr His Cys Ala Gly Thr 1 5 10
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Paecilomyces lilacinus
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 1..12
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Asp Gly Asn Gly His Gly Thr His Cys Ala Gly Thr 1 5 10
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Paecilomyces lilacinus
(ix) FEATURE:
(A) NAME/KEY: 3'clip
(B) LOCATION: 1..26
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GCNTAYACNC ARCARCCNGG NGCNCC 26
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Paecilomyces lilacinus
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION: 1..26
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GTNCCNGCRC ARRGNGTNCC RTGNCCRTTN CC 32

Claims

Claims
1. A protease from Paecilomyces lilacinus having a molecular weight of 20,00 to 200,000 Dalton an isoelectric point at a pH between 8 and 12, a pH optimum in a range from 9 and 12 and an enzymatic activity for surface structures of plant parasitic nematodes.
2. A protease according to claim 1 having a molecular weight of 25,000 to 100,000 Dalton.
3. A protease acoording to claim 1 having an isoelectric point which is at a pH of 9 to 11.
4. A protease according to claim 1 having an temperature optimum between 40 and 80 °C, preferably between 50 and 70 °C, especially between 55 and 65 °C.
5. A process for the preparation of the protease according to claim 1 , which comprises cultivation of Paecilomyces lilacinus and isolation of said protease.
6. The use of a fungal protease for the control of plant parasitic nematodes.
7. A gene for a protease having the DNA sequence shown in sequence protocol 1.
8. A plasmid containing a gene as claimed in claim 7.
9. A microorganism containg a gene as claimed in claim 7.
10. A plant or a plant cell containing a gene as claimed in claim 7.
PCT/EP1994/001395 1993-05-04 1994-05-02 New alkaline serine protease of paecilomyces lilacinus WO1994025579A2 (en)

Priority Applications (2)

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EP94916184A EP0700432A1 (en) 1993-05-04 1994-05-02 New alkaline serine protease of paecilomyces lilacinus
AU67949/94A AU6794994A (en) 1993-05-04 1994-05-02 New alkaline serine protease of paecilomyces lilacinus

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EP93107233A EP0623672A1 (en) 1993-05-04 1993-05-04 New alkaline serine protease of Paecilomyces lilacinus
EP93107233.4 1993-05-04

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WO1994025579A3 WO1994025579A3 (en) 1994-12-22

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US6187739B1 (en) 1995-09-21 2001-02-13 Henkel Kommanditgesellschaft Auf Aktien Paste-form washing and cleaning agents
US6248708B1 (en) 1996-09-05 2001-06-19 Henkel-Ecolab Gmbh & Co. Ohg Paste-form detergent containing a mixture of ethoxylated alcohols
US6329333B1 (en) 1997-01-30 2001-12-11 Henkel-Ecolab Gmbh & Co. Ohg Pastelike detergent and cleaning agent
US6500617B1 (en) 1998-05-01 2002-12-31 Maxygen, Inc. Optimization of pest resistance genes using DNA shuffling
US6627592B1 (en) 1998-12-15 2003-09-30 Ecolab Gmbh & Co. Ohg Pasty washing agent
WO2012171914A1 (en) 2011-06-14 2012-12-20 Bayer Intellectual Property Gmbh Use of an enaminocarbonyl compound in combination with a biological control agent
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WO2013110594A1 (en) 2012-01-25 2013-08-01 Bayer Intellectual Property Gmbh Active compound combinations containing fluopyram and biological control agent
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Cited By (15)

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Publication number Priority date Publication date Assignee Title
US6187739B1 (en) 1995-09-21 2001-02-13 Henkel Kommanditgesellschaft Auf Aktien Paste-form washing and cleaning agents
US6248708B1 (en) 1996-09-05 2001-06-19 Henkel-Ecolab Gmbh & Co. Ohg Paste-form detergent containing a mixture of ethoxylated alcohols
US6329333B1 (en) 1997-01-30 2001-12-11 Henkel-Ecolab Gmbh & Co. Ohg Pastelike detergent and cleaning agent
US6500617B1 (en) 1998-05-01 2002-12-31 Maxygen, Inc. Optimization of pest resistance genes using DNA shuffling
US6627592B1 (en) 1998-12-15 2003-09-30 Ecolab Gmbh & Co. Ohg Pasty washing agent
US9241493B2 (en) 2011-06-14 2016-01-26 Bayer Intellectual Property Gmbh Use of an enaminocarbonyl compound in combination with a biological control agent
WO2012171914A1 (en) 2011-06-14 2012-12-20 Bayer Intellectual Property Gmbh Use of an enaminocarbonyl compound in combination with a biological control agent
EP2540165A1 (en) 2011-06-30 2013-01-02 Bayer CropScience AG Use of a halogenated pesticide in combination with a biological pest control agent
WO2013092516A1 (en) 2011-12-19 2013-06-27 Bayer Intellectual Property Gmbh Use of an anthranilic diamide derivatives with heteroaromatic and heterocyclic substituents in combination with a biological control agent
EP2606732A1 (en) 2011-12-19 2013-06-26 Bayer CropScience AG Use of an anthranilic diamide derivatives with heteroaromatic and heterocyclic substituents in combination with a biological control agent
WO2013110591A1 (en) 2012-01-25 2013-08-01 Bayer Intellectual Property Gmbh Active compounds combination containing fluopyram bacillus and biologically control agent
WO2013110594A1 (en) 2012-01-25 2013-08-01 Bayer Intellectual Property Gmbh Active compound combinations containing fluopyram and biological control agent
US9730455B2 (en) 2012-12-03 2017-08-15 Bayer Cropscience Ag Composition comprising a biological control agent and an insecticide
CN106755170A (en) * 2016-12-15 2017-05-31 常熟浸大科技有限公司 A kind of preparation method of cytimidine and guanine
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