US20170204147A1 - Bioactive Protein, Use Thereof and Method for its Production - Google Patents

Bioactive Protein, Use Thereof and Method for its Production Download PDF

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US20170204147A1
US20170204147A1 US15/120,456 US201515120456A US2017204147A1 US 20170204147 A1 US20170204147 A1 US 20170204147A1 US 201515120456 A US201515120456 A US 201515120456A US 2017204147 A1 US2017204147 A1 US 2017204147A1
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plant
protein
trichoderma
hytlo1
longibrachiatum
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Matteo Lorito
Michelina Ruocco
Francesco Vinale
Sheridan Lois Woo
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Koppert BV
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Koppert BV
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • A01N63/04—
    • 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

  • the present invention relates to the field of agronomy. More in particular the invention relates to the stimulation and promotion of plant growth and/or plant health via the use of certain bioactive peptides alone and/or in combination with phytostimulants. According to certain aspects of the invention the promotion of plant growth and/or plant health is mediated by facilitating the interaction between the plant and plant stimulatory fungal species, such as Trichoderma spp.
  • Trichoderma spp. Fungi belonging to the genus Trichoderma are more and more subject of study, both because of their relevance as pathogen antagonists and their active and positive interaction with the colonized plants. Thanks to these beneficial characteristics, bio-pesticides and bio-fertilizer that use Trichoderma spp. as active ingredient are developed and commercialized (Harman et al., 2010, Plant Disease 94:928-939).
  • Trichoderma antagonistic activity is principally based on mycoparasitism (Lorito et al.,1993, Phytopathology, 83:313-318; Lorito et al.,1996, Molecular Plant-Microbe Interactions, 9:206-213; Sanz et al., 2004, Current Genetics, 46: 277-286; Zeilinger et al., 1999, Fungal Genetics and Biology, 26:131-140), antibiosis (Schirmbock et al., 1994, Applied and Environmental Microbiology, 60:4364-4370; Vinale et al., 2008, Physiological and Molecular Plant Pathology, 72: 80-86, Vinale et al., 2009, Letters in Applied Microbiology, 48, 705-711) and competition for nutrients and ecological niches (Vargas et al., 2009, Plant Physiology,151: 792-808).
  • Trichoderma strains can activate ISR (Hanson and Howell 2004, Phytopathology, 94:171-176; Mandal and Mitra, 2007, Physiological and Molecular Plant Pathology, 71: 201-209; Segarra et al. 2007, Proteomics, 7:3943-3952), a mechanism triggered upon root colonisation by non-pathogenic rhizobacteria or fungi and regulated by a specific signal transduction cascade (Pieterse et al., 1996, Plant Cell, 8:1225-1237; Segarra, et al., 2009, Plant Biology, 11:90-96).
  • plants whose roots are colonized by selected Trichoderma isolates are “sensitized” and respond faster and/or more intensely to pathogen attack, following a mechanism known as priming (Ahn, et al., 2007, Molecular Plant Pathogen Interaction, 20-759-768; Conrath et al., 2006, Plant Signalling and Behavior, 1:179-184).
  • the inventors of the present invention carried out experiments wherein the presence of Microbial Associated Molecular Patters proteins (MAMPs) produced by selected Trichoderma spp. during their interaction with plant material was investigated. Instead of selecting the Trichoderma species T. harzianum, T. atroviride, T. asperellum, T. virens most commonly applied in bio-pesticides and bio-fertilizer, the inventors of the present invention investigated a newly isolated strain of T longibrachiatum ( T. longibrachiatum strain MK1 (KV966), hereafter also indicated as MK1 or KV966. T.
  • MAMPs Microbial Associated Molecular Patters proteins
  • T. longibrachiatum MK1 incubations an about 7 KDa protein was selected for further analysis.
  • This protein turned out to be a Type II hydrophobin protein and thus was designated HYTLO1.
  • plant growth modulation including improvement of plant fresh weight, improvement of plant dry weight, improvement of root development, including improvement of root nodulation in Leguminosae, increase of germination percentage and increase of germination speed, disease and/or pathogen control and induction of systemic resistance.
  • Autologous proteins from Trichoderma species, in particular T. longibrachiatum having such features have not been described in the prior art.
  • the present invention thus according to a first aspect relates to the use of Trichoderma longibrachiatum , for the treatment of a plant, wherein the treatment of the plant preferably is aimed at one or more selected from(a) plant growth modulation, such as improvent of plant fresh weight, improvement of plant dry weight, improvement of root development, including improvement of root nodulation in Leguminosae, increase of germination percentage and/or increase of germination speed, or (b) disease control, such as via anti-microbial activity, preferably antifungal activity, or via stimulation of systemic resistance.
  • plant growth modulation such as improvent of plant fresh weight, improvement of plant dry weight, improvement of root development, including improvement of root nodulation in Leguminosae, increase of germination percentage and/or increase of germination speed
  • disease control such as via anti-microbial activity, preferably antifungal activity, or via stimulation of systemic resistance.
  • the Trichoderma longibrachiatum may be combined with the use of one or more plant biostimulants selected from (i) a Trichoderma species other than Trichoderma longibrachiatum , (ii) harzianic acid or a biologically active isomer thereof, such as iso-harzianic acid, (iii) 6-pentyl- ⁇ -pyrone or (iv) rhizobia.
  • a further aspect of the invention relates to a composition
  • a composition comprising a protein having at least 70% sequence similarity with an amino acid sequence according to SEQ ID NO: 1, characterized in that the protein is obtained as an autologous protein from Trichoderma longibrachiatum wherein in said composition the protein preferably is in a purified form.
  • the protein in the composition the protein may be combined with one or more plant stimulants, such as selected from (i) a Trichoderma species, preferably a Trichoderma species other than Trichoderma longibrachiatum , (ii) harzianic acid, or a biologically active isomer thereof, such as isoharzianic acid or (iii) 6-pentyl- ⁇ -pyrone.
  • Yet another aspect of the invention relates to the use of a protein having at least 70% sequence similarity with an amino acid sequence according to SEQ ID NO: 1, for the treatment of a plant, characterized in that the protein is provided as an autologous protein from Trichoderma longibrachiatum .
  • the treatment of the plant may be aimed at one or more selected from (a) plant growth modulation, such as improvement of plant fresh weight, improvement of plant dry weight, improvement of root development, including improvement of root nodulation in Leguminosae, increase of germination percentage and/or increase of germination speed, or (b) disease control, such as via antimicrobial activity, preferably antifungal activity, or via stimulation of systemic resistance.
  • the protein may be combined with the use of one or more plant biostimulants selected from (i) a Trichoderma species, preferably a Trichoderma species other than Trichoderma longibrachiatum , (ii) harzianic acid, or a biologically active isomer thereof, such as isoharzianic acid, (iii) 6-pentyl- ⁇ -pyrone or (iv) rhizobia.
  • a Trichoderma species preferably a Trichoderma species other than Trichoderma longibrachiatum
  • harzianic acid or a biologically active isomer thereof, such as isoharzianic acid, (iii) 6-pentyl- ⁇ -pyrone or (iv) rhizobia.
  • Further aspects of the invention relate to a method for producing a protein having at least 70% sequence similarity with an amino acid sequence according to SEQ ID NO: 1 and the protein obtainable with this method.
  • the method comprises:
  • the Trichoderma longibrachiatum strain MK1 (KV966) is yet a further aspect of the invention. This strain is suitable for application in the uses and methods of the invention. It has been deposited under the Budapest treaty with the Centraalbureau voor Schimmelcultures (CBS), Utrecht, the Netherlands, on Dec. 10, 2013 under deposit number CBS137023.
  • FIG. 1 Expression of Trichoderma longibrachiatum Hytlo 1 gene on salt medium (SMS)+1% (wt/vol) sucrose (lane 1), SMS+1% (wt/vol) cellulose (lane 2), SMS+1% (wt/vol) chitin (lane 3), SMS+1% (wt/vol) crude extract of tomato leaves (lane 4), SMS+1% (vol/vol) Rhizoctonia solani culture filtrate (lane 5), SMS+1% (wt/vol) R. solani purified cell walls (lane 6).
  • SMS salt medium
  • FIG. 2 SDS-PAGE silver stained of purified HYTLO1 (right lane) and molecular markers (left lane) in kD.
  • FIG. 3 HYTLO1 antifungal activity. Spore of the pathogens Botrytis cinerea and Alternaria spp. and of the biocontrol agent Trichoderma harzianum strain MK1-KV966 were germinated at 25° C. for 24 hours in Potato Dextrose Broth (PDB) containing 5 ⁇ M of HYTLO1 and compared with those germinated in PDB plus the same solvent as the control.
  • PDB Potato Dextrose Broth
  • FIG. 5 Effect of purified HYTLO1 (5 ⁇ M injected on tomato leaves after 48 hours under UV light. Fluorescence is due to accumulation of compounds related to plant defense response.
  • Lyophilized plant tissues (10 mg) from HYTLO1-injected and-non injected tomato leaves were collected at different time points after inoculation (from 6 to 48 h) and homogenized.
  • Anion superoxide was detected by measuring the reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamine) carbonyl]-2H-tetrazolium hydroxide (XTT) to formazan, according to Sutherland, M. W., and B. A. Learmonth, 1997, Free Radical Research, 27:283-289, with modifications reported by Castoria, R., et al., 2003, Phytopathology, 93:564-572.
  • ROOH Total hydroperoxides
  • FIG. 7 Effect of purified HYTLO1 on the activation of tomato plant defense responses. Leaves were injected with HYTLO1 at the indicated concentrations and analyzed after 48 hours by Real Time PCR for the accumulation of PR (Pathogenesis-Related)-proteins PR1, PR2 and PIN2 RNA (for methods see Tucci M., et al., 2011, Molecular Plant Pathology, 12:341-354) Values indicated the n. of fold increase vs. untreated control.
  • FIG. 8 Effect of purified HYTLO1 application on the infection of tomato leaves by the pathogen B. cinerea. 10 ⁇ L of a solution of HYTLO1 were applied to tomato leaves and 2 hours later 10 ⁇ L of a B. cinerea spore suspension 1 ⁇ 10 6 /ml were applied on the same spot. The measures were taken 72 hours post inoculation. The standard deviation bars were obtained from at least three different experiments, with five replicates and a total of 15 treated leaves per treatment.
  • FIG. 9 Effect of purified HYTLO1 application on tomato plant and root development. Tomato roots (left) and tomato cuttings (right) were dipped in a HYTLO1 solution of 0.018 ⁇ M.
  • FIG. 10 Effect of HYTLO1 on tomato plant growth as compared to treatment with T. harzianum strain T22 or the Commercial product GERMON E (G-E) base on the auxin 1-naphthaleneacetic acid, commercialized by L. Gobbi, Italy.
  • A Growth increase of root and stem of HYTLO1 vs. water control, GERMON E or T22, 10 days after treatment.
  • B Increase of root development (HYTLO1 treatment on the left, untreated control on the right),
  • C Effect of HYTLO1 on plant size compared to T22.
  • D Effect of HYTLO1 on root development compared to water control.
  • HYTLO1 was applied at final concentration in the soil at 10 ⁇ 8 M.
  • FIG. 11 Effect of purified HYTLO1 on tomato plants, applied by drench on the roots or by spraying on the leaves.
  • 50 ml of a solution containing 0.072 ⁇ g/ml of HYTLO1 (0.01 ⁇ M) were applied for each pot containing 1 litre of soil and 2 tomato plants, for a total of 4 applications.
  • 20 ml of a solution containing 0.072 ⁇ g/ml (0.01 ⁇ M) of HYTLO1 were sprayed for each pot containing 2 tomato plants for a total of 4 applications.
  • FIG. 12 Effect of purified HYTLO1 on the growth of Arabidopsis (A), percentage seed germination of cucumber (B) and plantlet development of Lotus japonicus (C).
  • A Arabidopsis thaliana (Columbia-0: Col-0) seeds were sown in Levington's F2 compost plus sand (JFC Munro, Devon; http://www.jfcmonro.com) and chilled for 2 days at 4° C. Plants were grown under short-day conditions (10 h light) in a controlled environment chamber, at 22° C. during the day and 18° C. at night with 60% relative humidity for 5-6 weeks before transplanting.
  • HYTLO1 Ten ml of sterile solutions of HYTLO1 were drenched every two days at concentrations 0.072 ⁇ g/ml (0.01 ⁇ M) for one month in each pot containing 100 ml of soil and 1 Arabidopsis plant. Untreated plants were used as controls.
  • B Sterile cucumber seeds were placed on magenta box containing half-strength Murashige and Skoog salt (MS) medium (ICN Biomedicals) containing 1% agar and 1.5% sucrose, adjusted to pH 5.7, and vernalized for 2 days at 4° C. in the absence of light.
  • a sterile solutions of HYTLO1 was added to the substrate at final concentration of 10 ⁇ 8 M before the solidification of agar.
  • C The same concentration of HYTLO1 was used in the agarized medium used for the L. japonicus test.
  • FIG. 13 Effect of transgenic expression of the Hytlo1 gene on tomato. Cuttings quickly form new roots in the presence of water.
  • FIG. 15 Effect of Hytlo1 knock-out on the in vivo biocontrol activity of T. longibrachiatum .
  • the wild-type strain (Wt), 1 representative ⁇ Hytra1 mutant (T3) and an empty vector-transformed control were applied as a seed coating.
  • Untreated controls (T ⁇ ) and Trichoderma -treated tomato plants were infected with B. cinerea by inoculating the third true leaf with 10 ⁇ L of a 1 ⁇ 10 6 /ml spore suspension of the pathogen. Inoculated plants were enveloped in transparent plastic bags to achieve high relative humidity conditions and incubated in a growth chamber at 18° C. with a photoperiod of 16 h light.
  • the disease spread was recorded at 2 (2 dpi), 3 (3 dpi) and 5 (5 dpi) days post infection, by measuring necrotic lesions with an electronic calliper and calculating the area as an ellipse. Values are means with SD obtained from at least three different experiments with five replicates and a total of 15 treated plants per treatment.
  • FIG. 16 Effects of Hytlo1 gene knock-out on the plant growth promotion activity of T. longibrachiatum strain MK1-KV966 (A, effect on the roots; B, effect on the entire plant).
  • Trichoderma seed treatments were: untreated control (T ⁇ ), wild type T. longibrachiatum (wt); Hytlo1 mutants (T1, T3, T4), empty vector control (Thph). Bars indicate standard deviation from three experiments with four replicates per treatment.
  • FIG. 18 Enhancement of nodule formation by treatment of R. lotii acting on L. japonicus by adding different metabolites from Trichoderma spp.
  • Wt control with R. loti without metabolites.
  • the purified molecules were added to the sterile MS media and poured into Petri dishes. Average number of nodules per cm of root are indicated. Data were recorded 4 weeks after seeding of L. japonicus.
  • Trichoderma species are known for their beneficial effects on plant health and plant growth. However, the recorded benefits of Trichoderma species is mostly restricted to T. harzianum, T. virens , T asperellum and T. atroviride. T longibrachiatum in general is less considered in association with beneficial effects on plants or for use as biocontrol agent. Instead this species is scientifically described and commercially used in relation to the production of plant biomass degrading enzymes. The inventors of the present invention however have surprisingly found that T. longibrachiatum is able to produce a bioactive hydrophobin, designated HYTLO1, that has a distinct positive effects on plants. In addition HYTLO1 has negative effects on various plant pathogens. Thus HYTLO1 may be used in the treatment of plants. Positive effects of T. longibrachiatum on plants may be selected from plant growth modulation and/or disease control, including disease prevention, and/or stimulation of systemic resistance.
  • Modulation should be understood as any change or alteration including alterations that can be considered as improvements (increases), but also alterations that may be considered as reductions (decreases). Modulations that can be considered as improvements or increases (stimulations) are preferred.
  • plant growth modulation may be understood as referring to modulation of growth parameters of plants.
  • the modulation of plant growth parameters may be established with reference to an untreated control.
  • the plant growth parameter may be root development. Root development may be assessed with any method known to the skilled person, for example as exemplified in the examples.
  • the plant growth modulation is improvement (stimulation) of root nodulation in a plant from the family Leguminosae (or alternatively named Fabaceae).
  • Root nodules are root organs of Leguminosae containing symbiotic rhizobia capable of fixing atmospheric nitrogen.
  • Root nodulation is the process of root nodule formation.
  • the plant from the family Leguminosae may be selected as presented previously.
  • Glycine max is selected with particular preference in connection to the stimulation of root nodulation.
  • Stimulation (improvement) of nodulation may be an increase of the nodule density and may be assessed on the basis of this, as is exemplified in the examples.
  • An alternative plant growth parameter that may be modulated is the germination percentage.
  • the germination percentage may be assessed with any method known to the skilled person, for example by using an hemocytometer
  • the germination speed may be assessed with any method known to the skilled person, for example by using an hemocytometer.
  • T. longibrachiatum in addition to modulation of plant growth also has beneficial effects on disease control.
  • Disease control should be understood as meaning to include both disease prevention and/or treatment of a disease.
  • Diseases that may be effectively controlled with T. longibrachiatum include, but are not restricted to fungal diseases, such as fungal diseases caused by soil-borne fungal pathogens such as Rhizoctonia spp., for example Rhizoctonia solani, Fusarium spp. Fusarium graminearum, Pythium spp. for example Pythium ultimum , or fungal diseases caused by aerial-borne fungal pathogens such as Alternaria spp. Alternaria alternata, Botrytis spp. for example Botrytis cinerea , but also diseases caused by bacterial pathogens such as Xantomonas spp. for example Xantomonas campestris.
  • ISR Induced systemic resistance
  • plants respond with a signalling cascade that leads to the systemic expression of a broad spectrum and long lasting disease resistance that is efficient against amongst others fungi, bacteria and viruses.
  • Induced systemic resistance is amongst others associated with the activation of plant defence responses, which may include “oxidative burst ”, synthesis of phytoalexins, accumulation of PR (Pathogenesis-Related) proteins, localized programmed cell death also known as HR (Hypersensitive Response), etc.
  • the plant selected for treatment may be any plant, in particular a plant from a commercially relevant crop.
  • the plant may be selected as any plant in a particular commercially relevant plant, such as a plant of a food crop, an ornamental plant, a flower plant.
  • the plant may be selected from the family Solanaceae , in particular from the subfamily Solanoideae , such as from the genus Solanum , for example Solanum lycopersicum (tomato), Solanum melongena (egg plant), Solanum tuberosum (potato), from the genus Capsicum , such as Capsicum annuum or Capsicum frutescens , from the genus Physalis such as physalis philadelphica (tomatillo), but also from the genus Petunia , the genus Browallia , the genus Lycyanthes or the subfamily Nicotianoideae such as from the Genus Nico
  • the skilled person will know that of the plant species mentioned above, various varieties are available for cultivation. Reference to a specific plant species is to be considered as including the various varieties available.
  • the selected plant may be in any developmental stage such as a seed, a seedling, a young developing plant, or a full grown plant.
  • T longibrachiatum on plants are linked to the HYTLO1 protein having the amino acid sequence of SEQ ID NO: 1.
  • This protein has been found in the secreotome of the T. longibrachiatum strain MK1.
  • additional T. longibrachiatum strains may express proteins having amino acid sequences similar to the HYTLO1 protein.
  • the invention in general relates to the use of Trichoderma longibrachiatum for the treatment of a plant.
  • the Trichoderma longibrachiatum is in particular from a strain comprising an autologous nucleotide sequence coding for a protein having at least 70% sequence similarity with the amino acid sequence of SEQ ID NO: 1, such as Trichoderma longibrachiatum strain MK1.
  • the nucleotide sequence coding for the amino acid sequence may be the sequence of SEQ ID NO: 2.
  • a nucleotide sequence coding for protein sequences may be a DNA sequence and/or a RNA sequence.
  • an autologous nucleotide sequence is a nucleotide sequence present in the autologous genome of an organism, i.e. it is not introduced from an external source viz. it is not a heterologous sequence.
  • the T. longibrachiatum used in the invention is not genetically modified in respect of the HYTLO1 protein.
  • the selected T. longibrachiatum does not comprise any artificially introduced foreign nucleotide sequences i.e. it is a non-GM organism.
  • the autologous nucleotide sequence codes for a protein having at least 70% sequence similarity with the amino acid sequence of SEQ ID NO: 1.
  • at least 70% similarity should be understood as meaning over 70%, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%. at least 99% sequence similarity.
  • sequence similarity is used it embraces sequence “identity” and “conservative changes”. According to certain embodiments the conservative changes are disregarded and the % sequence similarity refers to % sequence identity.
  • sequence identity is known to the skilled person.
  • sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • alignment may be carried out over the full lengths of the sequences being compared.
  • the alignment may be carried out over a shorter comparison length, for example over about 20, about 50, about 100 or more nucleic acids/bases or amino acids.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the two sequences being compared are of the same or substantially the same length.
  • the percentage of “conservative changes” may be determined similar to the percentage of sequence identity. However, in this case changes at a specific location of an amino acid or nucleotide sequence that are likely to preserve the functional properties of the original residue are scored as if no change occurred.
  • amino acid sequences are the physico-chemical properties of the amino acids.
  • a conservative substitution for an amino acid in a polypeptide of the invention may be selected from other members of the class to which the amino acid belongs.
  • an amino acid belonging to a grouping of amino acids having a particular size or characteristic can be substituted for another amino acid without substantially altering the activity of a protein, particularly in regions of the protein that are not directly associated with biological activity (see, e.g., Watson, et al., Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 -4th Edition 1987).
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine.
  • Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • Conservative substitutions include, for example, Lys for Arg and vice versa to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr and vice versa so that a free —OH is maintained; and Gln for Asn and vice versa to maintain a free —NH2.
  • nucleotide sequences the relevant functional property is mainly the biological information that a certain nucleotide carries within the open reading frame of the sequence in relation to the transcription and/or translation machinery. It is common knowledge that the genetic code has degeneracy (or redundancy) and that multiple codons may carry the same information in respect of the amino acid for which they code.
  • amino acid leucine is coded by UUA, UUG, CUU, CUC, CUA, CUG codons (or TTA, TTG, CTT, CTC, CTA, CTG for DNA), and the amino acid serine is specified by UCA, UCG, UCC, UCU, AGU, AGC (or TCA, TCG, TCC, TCT, AGT, AGC for DNA). Nucleotide changes that do not alter the translated information are considered conservative changes.
  • the skilled person will be aware of the fact that several different computer programs, using different mathematical algorithms, are available to determine the identity between two sequences. For instance, use can be made of a computer program employing the Needleman and Wunsch algorithm (Needleman and Wunsch, Journal of Molecular Biolology, 48, 443-453). According to an embodiment the computer program is the GAP program in the Accelrys GCG software package (Accelrys Inc., San Diego U.S.A). Substitution matrices that may be used are for example a BLOSUM 62 matrix or a PAM250 matrix, with a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the Accelrys GCG software package (Accelrys Inc., San Diego U.S.A)
  • Accelrys GCG software package Accelrys Inc., San Diego U.S.A
  • a NWSgapdna CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6 is used.
  • the percent identity of two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (Meyers and Miller, 1989. Bull. Math. Biol. 51, 5-37) which has been incorporated into the ALIGN program (version 2.0) (available at the ALIGN Query using sequence data of the Genestream server IGH Montpellier France http://vegajgh.mrs.fr/bin align-guess.cgi) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • BLAST Basic Local Alignment Tool
  • BLAST queries are performed with the following parameters.
  • the percentage of “conservative changes” may be determined similar to the percentage of sequence identity with the aid of the indicated algorithms and computer programs.
  • Some computer programs, e.g., BLASTp, present the number/percentage of positives ( similarity) and the number/percentage of identity.
  • Trichoderma longibrachiatum is combined with the use of one or more plant biostimulants selected from (i) a Trichoderma species other than Trichoderma longibrachiatum , (ii) harzianic acid or a biologically active isomer thereof, such as, iso-harzianic acid, (iii) 6-pentyl- ⁇ -pyrone or (iv) rhizobia.
  • HA, iso-HA and 6PP are known metabolites of Trichoderma species having beneficial effects on plants.
  • such phytostimulatory agents provide additional effects on plants in respect of (a) plant growth modulation, such as improvement of plant fresh weight, improvement of plant dry weight, improvement of root development, including improved nodulation in Leguminosae, increase of germination percentage and/or increase of germination speed, or (b) disease control such as via anti-microbial activity, preferably antifungal activity, or via stimulation of systemic resistance.
  • plant growth modulation such as improvement of plant fresh weight, improvement of plant dry weight, improvement of root development, including improved nodulation in Leguminosae, increase of germination percentage and/or increase of germination speed
  • disease control such as via anti-microbial activity, preferably antifungal activity, or via stimulation of systemic resistance.
  • Trichoderma species different from Trichoderma longibrachiatum having plant stimulatory features may be readily identified by the skilled person and may for example be selected from T. harzianum (for example strain T22), T. atroviride (for example strain P1), T asperellum, T. virens (for example strain GV41).
  • Harzianic acid (CAS: 157148-06-6) or alternatively HA is an alkaloid compound of the structural formula (1) below, having the chemical formula C 19 H 27 NO 6 and a MW of 365.42.
  • HA is commercially marketed by various parties and may amongst others be obtained in a suitable form from CHEMFACES (http://www.chemfaces.com/natural/Harzianic-acid-CFN00118.html).
  • harzianic acid includes all stereochemical isomers of the compound of formula 1, such as iso-harzianic acid.
  • Iso-Harzianic acid or alternatively iso-HA is a stereoisomer of HA with the structural formula (2) below, having the chemical formula C 19 H 27 NO 6 and a MW of 365,42.
  • 6-Pentyl-a-pyrone (CAS: 27593-23-3) or alternatively 6PP is a compound of the structural formula (3) below having the chemical formula C 10 H 14 O 2 and a MW of 166.216. It is commercially marketed by various parties and may amongst others be obtained in a suitable form from SIGMA.
  • Rhizobia are nitrogen-fixing bacteria capable of living in a symbiotic relationship with plants from the family Leguminosae .
  • the skilled person will know that rhizobia may have a certain host specificity and it is within the ambit of the knowledge of the skilled person to select rhizobia capable of living in a symbiotic relationship with a selected plant from the family Leguminosae .
  • any rhizobia may be used that have specificity for a selected plant from the family Leguminosae , such as from the genus Glycine , for example Glycine max (soy bean), from the genus Phaseolus , from the genus Pisum , for example Pisum sativum (pea), from the genus Cicer , for example Cicer arietinum , from the genus Medicago , for example Medicago sativa (alfafa), from the genus Arachis , for example Arachis hypogaea (peanut), from the genus Ceratonia , for example Ceratonia siliqua (carob), from the genus Glycyrrhiza , for example Glycyrrhiza glabra (liquorice), or from the genus Lupinus , for example Lupinus angustifolius .
  • Glycine for example Glycine max (soy bean)
  • the rhizobia for example may be selected from the genus Rhizobium such as R. leguminosarum, R. tropici, R. loti, R. trifolii, R. meliloti, R. fredii, R. cellulosilyticum, R. daejeonense, R. etli, R. galegae, R. gallicum, R. giardinii, R. hainanense, R. huautlense, R. indigoferae, R. loessense o huanglingense, R. lusitanum, R. mongolense, R. sullae o hedysari, R.
  • Rhizobium such as R. leguminosarum, R. tropici, R. loti, R. trifolii, R. meliloti, R. fredii, R. cellulosilyticum, R. daejeonense, R.
  • Rhizobium mediterraneum M. plurifarium, M. septentrionale, M. temperatum, M. tianshanense (ex Rhizobium tianshanense ), or from the genus Azorhizobium such as A. caulinodans, A. doebereinerae o johannae or from the genus Methylobacterium such as M. nodulans or from the genus Ensifer (ex Sinorhizobium ) such as E. abri, E. americanum, E. arboris, E. fredii (ex Rhizobium fredii ), E. indiaense, E. kostiense, E.
  • selected rhizobia preferably are “soybean rhizobia” capable of forming root nodules in soybean ( Glycine max ), such as for example Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium diazoefficiens, Bradyrhizobium liaoningense, Ensifer fredii, Ensifer xinjiangenese, Mesorhizobium tianshanense or others known to the skilled person.
  • a further aspect of the invention relates to a composition
  • a composition comprising a carrier and a source of protein having at least 70% sequence similarity with an amino acid sequence according to SEQ ID NO: 1.
  • the composition is characterized in that the protein is an autologous protein from Trichoderma longibrachiatum.
  • the composition preferably is an agronomical composition, for example an agronomical composition aimed at one or more selected from plant growth modulation, such as improvent of plant fresh weight, improvement of plant dry weight, improvement of root development, including improvement of root nodulation in Leguminosae, increase of germination percentage and/or increase of germination speed, or disease control or stimulation of systemic resistance.
  • the carrier may be selected from any suitable carrier, such as an agronomically acceptable carrier. Selection of suitable carriers is within the ambit of the knowledge of the skilled person. Suitable carriers or co-formulants could be clay, sands, active carbon, for solid formulations or lactose/celluloses for immobilization and dispersal of solid and liquid formulations.
  • HYTLO1 and HA could be dissolved in ethanol or methanol at low concentration 10 ⁇ 3 M and diluted in water at 10 ⁇ 5 to10 ⁇ 6 M for the application.
  • 6PP could be dissolved in ethyl acetate or acetone at 10 ⁇ 3 M to be further dissolved in water to 10 ⁇ 5 to10 ⁇ 6 M for the application.
  • Anti-oxidants such as benzotriazole, benzophenone, acrylate could be added to the composition to increase the shelf-life.
  • Liquid formulations could be added to a solid, spore-based formulation of living Trichoderma or other microbe to potentiate the effect.
  • the active principle could be prepared by freeze drying, spray drying, fluid-bed drying, vacuum drying, tray drying or liquid stabilisation by buffering the pH.
  • the designing of the final formulation could be done, but not limited to, by processing the pure or crude protein in blended mixed in powders or dissolved in liquids or solvents.
  • formulations will be optimized so that an amount of 30 ml (30 gr if dried) to 5 Liter (5 kg if dried) of formulated product will be applied for 1 hectare of crop.
  • Application to the crop may be by any known means such as by using drip irrigation systems, drenching, fogging or spraying or any other crop application system.
  • a source of the protein should be understood as a source suitable to provide the protein.
  • the source of the protein may be biomass of a Trichoderma longibrachiatum strain, such as Trichoderma longibrachiatum strain MK1 deposited as CBS 137023, preferably viable biomass.
  • the source is selected as viable biomass the protein need not be present but instead may be expressed in situ at a later stage. This is in particular the case when viable spores are selected as the biomass.
  • the source of the protein may be a product derived from such biomass, such as an at least partially purifed extract.
  • the protein preferably is provided from a source wherein it is in a purified state or form.
  • a purified state or form should be understood as a state wherein the protein is more pure than in its original state, i.e. it is cleaned and/or separated from foreign elements and/or elements present in the environment from which it originates.
  • the protein need not be 100% pure. Instead it may have a purity of for example 40-99.9%, such as 50-99%, or 60-95%, or 70-90%, or 80-90%, or 85-90%.
  • the content of the protein in the composition may be between 100% w/w (pure protein) and 0.1 ppb (1 ⁇ 10 ⁇ 10 ) or , such as between 10% w/w and 0.1 ppb, between 5% w/w and 1 ppb, between 1% w/w and 5 ppb, between 0.1% w/w and 10 ppb, based on the activity demonstrated in our assays.
  • the protein may be combined with one or more plant biostimulants such as selected from (i) a Trichoderma species, preferably a Trichoderm species other than Trichoderma longibrachiatum , (ii) harzianic acid, or a biologically active isomer thereof, such as iso-harzianic acid or (iii) 6-pentyl- ⁇ -pyrone.
  • plant biostimulants such as selected from (i) a Trichoderma species, preferably a Trichoderm species other than Trichoderma longibrachiatum , (ii) harzianic acid, or a biologically active isomer thereof, such as iso-harzianic acid or (iii) 6-pentyl- ⁇ -pyrone.
  • a Trichoderma species preferably a Trichoderm species other than Trichoderma longibrachiatum
  • harzianic acid or a biologically active isomer thereof, such as iso-harzianic acid or (iii)
  • a further aspect of the invention relates to the use of a protein having at least 70% sequence similarity with an amino acid sequence according to SEQ ID NO: 1, for the treatment of a plant.
  • This aspect of the invention is characterized in that the protein is provided as an autologous protein from Trichoderma longibrachiatum .
  • the use of the protein is combined with the use of one or more plant biostimulants such as selected from (i) a Trichoderma species, preferably a Trichoderma species other than Trichoderma longibrachiatum , (ii) harzianic acid, or a biologically active isomer thereof, such as iso-harzianic acid or (iii) 6-pentyl- ⁇ -pyrone.
  • the treatment of the plant is aimed at one or more selected from (a) plant growth modulation, such as improvement of plant fresh weight, improvement of plant dry weight, improvement of root development, including improvement of root nodulation in Leguminosae, increase of germination percentage and/or increase of germination speed, or (b) disease control, such as via anti-microbial activity, preferably antifungal activity, or via stimulation of systemic resistance.
  • plant growth modulation such as improvement of plant fresh weight, improvement of plant dry weight, improvement of root development, including improvement of root nodulation in Leguminosae, increase of germination percentage and/or increase of germination speed
  • disease control such as via anti-microbial activity, preferably antifungal activity, or via stimulation of systemic resistance.
  • a further aspect of the invention relates to a method for producing a protein having at least 70% sequence similarity with an amino acid sequence according to SEQ ID NO: 1.
  • the method comprises:
  • Trichoderma longibrachiatum comprising a functional autologous gene coding for the protein
  • Trichoderma longibrachiatum comprising a functional autologous gene coding for the protein having at least 70% sequence similarity with an amino acid sequence according to SEQ ID NO: 1 is provided.
  • the skilled person is able to determine whether or not a certain T. longibrachiatum strain comprises a functional autologous gene coding for the protein.
  • T. longibrachiatum strain MK1 may be selected.
  • the non-GM T may be selected.
  • longibrachiatum strain and any protein isolated therefrom any nucleotide sequences present will be free of nucleotide sequences from a different organism.
  • products obtained by the use of genetic modification are received with reservations and even banned by certain consumer groups.
  • special regulations may apply to bringing GM organisms and products derived therefrom in the environment. In view of this, non-GM production has certain benefits.
  • T. longibrachiatum may be provided in any suitable viable form, such as in the form of spores and/or hyphal biomass.
  • biomass of T. longibrachiatum is cultured under conditions suitable for expression of the gene.
  • Culturing may be in a solid state fermentation or a liquid fermentation or semi-solid fermentation.
  • the skilled person will be able to determine suitable culture conditions for both solid state fermentations and liquid fermentation to allow growth of T. longibrachiatum and expression of the gene.
  • T. longibrachiatum may be grown on Murashige and Skoog medium+0.8-1.3%, such as 1% sucrose for 6-8, such as 7 days at 23-27° C., such as 25° C. under aeration e.g. by shaking at 150 rpm.
  • the protein is obtained.
  • the protein may be obtained in the from of biomass, preferably viable biomass.
  • the protein may be isolated from the biomass and/or the culture broth . It is believed that the protein is secreted and is also bound to the fungus structure lining the external surface. Methods for isolation/purification of proteins from cultured biomass are known and/or can be easily developed by the skilled person without an undue burden.
  • the methods presented in example 3 may be used.
  • Other methods based on chromatography techniques that exploit the size or the hydrophobic properties of the proteins may be used.
  • a method to obtain the protein could be either by membrane purification, starting from centrifugating the biomass followed by fractionation of the supernatant fractionation by a membrane filter with a final cutting of 5 kD, thus obtaining the about 7 kD protein.
  • Another method could be based on adsorption chromatography or expanded bed chromatography (Birger et al., 1999, Journal of Chromatography A, 865:129-144). These methods could be integrated by extraction with an appropriated solvent for hydrophobic compounds.
  • a further aspect of the invention relates to the protein obtainable from the method for producing a protein having at least 70% sequence similarity with an amino acid sequence according to SEQ ID NO: 1.
  • This protein is produced as an autologous protein and may thus may be considered to be a non-GM product, in particular when produced in a T. longibrachiatum strain devoid of foreign (heterologous) DNA i.e. a non-GM strain.
  • the invention relates to Trichoderma longibrachiatum strain MK1 (KV966).
  • This strain comprises an autologous nucleotide sequence coding for a protein having at least 70% sequence similarity with the amino acid sequence of SEQ ID NO: 1 and thus may be used as a source for obtaining this protein, in particular as a non-GM product. It has been deposited with The Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands (CBS) on Dec. 10, 2013 under deposit number CBS 137023. The strain was isolated from natural soil in an agricultural field and it was identified as Trichoderma longibrachiatum on the basis of morphological and physiological characteristics.
  • Trichoderma longibrachiatum strain MK1 may be cultured with methods suitable for culturing other Trichoderma longibrachiatum strains known to the skilled person, e.g. by using the culture conditions as detailed above and as exemplified in experiment 3.
  • reference to Trichoderma longibrachiatum strain MK1 refers to the strain deposited under deposit number CBS 137023 or any equivalent strain, including derived strains, having its characterising features.
  • Seeds treated with a source of a protein having at least 70% sequence similarity with an amino acid sequence according to SEQ ID NO: 1, said protein being obtained as an autologous protein from Trichoderma longibrachiatum are yet a further aspect of the invention.
  • the seed may be from any plant, preferably from a commercially relevant plant as identified above.
  • a further aspect of the invention relates to a method for producing crop plants which employs the use of a source of the HYTLO1 protein.
  • the method comprises the steps of:
  • a number of plants is provided.
  • a number of should be understood as meaning one or more and includes in particular a plurality.
  • the plants may be selected as any plant in particular commercially relevant plants as identified above. Again, the skilled person will know that of the plant species mentioned above, various varieties are available for cultivation. Reference to a specific plant species is to be considered as including the various varieties available.
  • the plants may be provided as seeds, seedlings or young developing plants.
  • the provided number of plants is treated with a source of the HYTLO1 protein.
  • Treatment may comprise contacting the plant with the protein source.
  • seeds, seedlings of developing plants may be contacted with the protein source by applying a liquid solution comprising the protein source.
  • the liquid solution may be applied by means of soaking as a drench, by watering or by spraying.
  • the liquid solution may have a content of the protein ranging from pure protein to 1 ⁇ 10 ⁇ 11 M, such as 50% w/v to 1 ⁇ 10 ⁇ 9 M, preferably 1 ⁇ 10 ⁇ 3 M to 1 ⁇ 10 ⁇ 9 M, such as 1 ⁇ 10 ⁇ 5 M to 1 ⁇ 10 ⁇ 8 M, more preferably 5 ⁇ 10 ⁇ 6 M to 1 ⁇ 10 ⁇ 7 M.
  • the number of plants is provided with suitable nutrients and environmental conditions to allow the plant to develop further.
  • suitable nutrients such as N, P, S. CO 2
  • environmental conditions such as a suitable root substrate, suitable temperature and suitable light conditions
  • T. longibrachiatum low molecular weight secretome was analysed by SDS-PAGE.
  • a protein with an estimated molecular weight of about 8 kDa resulted to be profusely secreted when T. longibrachiatum was grown in liquid media added with plant tissue, fungal biomass or cellulose.
  • the band, corresponding to this protein, was excised from the SDS-PAGE gel and sequenced by Edman reaction process. The first 15 amino acidic residues of the protein were obtained. Blastp analysis of this peptide provides intermediate level homologies with hydrophobin from various fungi including T.
  • Hytlo1 cDNA sequence resulted 216 bp long and the in silico translation gave a predicted protein of 71 aa (SEQ ID NO: 1), with a molecular weight of 7218 Da and 8 cysteine residues arranged in the strictly conserved hydrophobin motif X n —C—X 5-10 —C—C—X 11-44 —C—X 8-23 —C—X 5-9 —C—C—X 6-18 —C—X m .
  • the genomic copy of the Hytlo1 gene was obtained by PCR amplification performed on T.
  • Hytlo1For GCTGTCTGCCCTACCGGCC
  • Hytlo 1Rev TTACTGGGTGCCGAGGGC
  • T. longibrachiatum Hytlo 1 Northern analysis indicated that the transcription of the T. longibrachiatum Hytlo 1 gene was strongly activated when T. longibrachiatum was grown in a basic salt medium (SMS) in the presence of sucrose, cellulose and chitin, and less in the presence of tomato leaf extracts, or R. solani culture filtrate or cell walls ( FIG. 1 ).
  • SMS basic salt medium
  • T. longibrachiatum MK1 spore suspension 10 8 /ml was used to inoculate Erlenmeyer flasks containing 100 ml of Murashige and Skoog (M&S base medium) enriched with 1% sucrose.
  • M&S base medium Murashige and Skoog
  • the culture filtrate (CF) was separated from the biomass by filtration with Miracloth paper (Calbiochem La Jolla, Calif. USA) and subsequently centrifugated at 20.000 rpm for 20 min.
  • the obtained clear CF was poured in a separator funnel, vigorously shaken for 5 minutes and decanted for 5 more minutes.
  • Purified HYTLO1 had direct inhibitory effect on germination of spores of Botrytis cinerea and Alternaria spp., but not of T. harzianum as shown in FIG. 3 .
  • T. longibrachiatum HYTLO1 Antifungal effect of T. longibrachiatum HYTLO1 was also tested in comparison with similar hydrophobins (of similar size) from two different Trichoderma strains, but only HYTLO1 from T. longibrachiatum strain MK1 exhibited a strong antifungal effect on a ELISA-based inhibition assay ( FIG. 4 ).
  • HYTLO1 In order to determine if purified HYTLO1 is able to protect the plant from foliar infection by a pathogenic fungus, the protein was tested on tomato grown-up plants against Botrytis cinerea . Indeed the application of any tested concentration of HYTLO1 above 1.25 ⁇ M (concentration of the HYTLO1 solution of which 10 ⁇ L were applied) significantly reduced the size of the developing necrotic area caused by the pathogen up to 72 hr after the inoculation ( FIG. 8 ). This demonstrate that HYTLO1 can be used directly as an antimicrobial agent for plant protection.
  • HYTLO1 Enhances Plant Growth and Development.
  • harzianum T22 (applied as spore suspension according the typical application rate of commercial products) and that of the germinating hormone 1-naphthaleneacetic acid (GERMON E-produced by L. Gobbi, Italy) (applied at the typical application rate of commercial products).
  • Applications were made once per week for one month by watering the tomato plants. Results, expressed in terms of plant height and length, root length and weight, number of leaves, stem diameter and weight, are combined and shown in FIG. 10 and FIG. 11 .
  • HYTLO1 treatment increased all of the growth parameters considered in comparison to any of the other treatments as well as of the untreated control.
  • the growth promotion effect of HYTLO1 apart from tomato (Solanaceae) was also confirmed on representative plant speciesof other plant families ( FIG.
  • HYTLO1 shows as examples the effect on Arabidopsis thaliana ( Brassicaceae ), Cucumis sativus ( Cucurbitaceae ), and Lotus japonicus ( Leguminosae )).
  • the experiments demonstrate that HYTLO1 can be effectively used to promote growth and productivity of a variety of crop plants, as well to stimulate seed germination, and root formation.
  • HYTLO1 has a Key Role of in the Biocontrol Activity of T. longibrachiatum
  • HYTLO1 The role of HYTLO1 on the biocontrol and plant growth promotion effect of Trichoderma longibrachiatum was demonstrated by targeted knock-out of the Hytlo1 gene by using a standard protoplast transformation method based on treatment with polyethylene glycol (PEG) was used (Punt et al., 1987, Gene, 56:117-124). The lack of Hytlo1 transcripts in a few randomly selected mutants was confirmed by RT-PCR and Northern analysis. The growth rate of the selected ⁇ -Hytlo1 mutants was the same as of wild type both on solid or liquid growth substrates, while spores were similar in colour, shape and number to those of the wild type. However, the selected mutants were strongly impaired when growing against R. solani or P.
  • HYTLO1 for plant growth promotion applied either singly or in combination with other beneficial molecules
  • Tested metabolites include 6PP and Harzianic Acid, and experiments were performed on seed germination, stem or root elongation as well fresh/dry weight.
  • the results shown in Table 1 were obtained by using cucumber seeds coated with the solutions of the tested compounds, that were then transferred in Petri dishes and left to germinate on filter humid paper. First seed germination was evaluated, then the germinated seedlings were transplanted into pots containing sterilised soil.
  • the purified protein was applied in combination with two strains of T. harzianum (T22 and TH1) and one strain of T. asperellum (CS1).
  • T. harzianum T22 and TH1
  • T. asperellum CS1
  • Table 2 expressed in terms of increased stem and root length or fresh/dry weight.
  • Cucumber seeds were coated with the purified molecule solutions and/or spore suspension, transferred into Petri dishes and left to germinate on humid filter paper. Germinated seedlings were transplanted into pots containing sterilised soil.
  • HYTLO1 either applied alone or in combination with HA or 6PP enhances the plant growth promotion effect of rhizobia, such as Rhizobium sp., on plant, and that this phenomenon may be due to an increased number of nodule formation on the treated roots.
  • This data demonstrate that HYTLO1 can be used to enhance the efficacy and usefulness of rhizobia-based, such as Rhizobium -based, microbial inocula, in terms of promotion of plant agronomic performance as well as in fertilizing the soil due to nitrogen fixation by the nodulated bacteria.

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