WO2019145693A1 - Inhibitory agent - Google Patents

Abstract

The disclosurerelates to an agent comprising inhibitory RNA, for example siRNA or antisense RNA and its use as an inhibitory agent for pathogenic eukaryotic microbial species such as pathogenic fungal species and oomycetes.

Description

Inhibitory Agent

Field of the Invention The disclosure relates to an agent comprising inhibitory RNA, for example siRNA or antisense RNA and its use as an inhibitory agent for pathogenic eukaryotic microbial species such as pathogenic fungal species and oomycetes.

Background to the Invention

Lytic polysaccharide monooxygenases (LPMOs) represent a group of enzymes which have been shown to play a key role in the breakdown of crystalline cellulose and chitin (20, 21 and 22). They achieve this by catalysing the oxidative cleavage of insoluble polysaccharides at the crystal surface using molecular oxygen and an electron donor. These so-called Auxiliary Activity (AA) enzymes dramatically boost the breakdown of complex carbohydrates by conventional glycoside hydrolases (20, 22, and 23) and have been included in commercial cocktails (24). Until now, all known classes of LPMOs come from bacteria, fungi and viruses (19). LPMOs are known in the art. WO2016142536 discloses LPMOs capable of cleaving cellulose and xylan. WO2015165951 discloses a process for the enzymatic hydrolysis of lignocellulosic material wherein said process comprises at least two cellulases and at least one LPMO. CN 104593279 discloses a genetically engineered bacterium containing a nucleic acid encoding a LPMO improving the generation of glucose when applied to raw materials such as cellulose. WO2015017869 discloses a variety of fungal LPMOs for use in a process for producing fermentable sugars from biomass.

LPMOs are also found in a variety of plant pathogens such as Botrytis, Phytophtora, Sclerotinia sclerotiorum and Hyaloperoospora.

Botrytis is a necrotrophic fungus and affects a variety of plant species such as strawberries or grapes resulting in rotting of fruits, flowers, leaves, storage organs or shoots. Oomycetes are fungus-like eukaryotic microorganism with saprophytic or pathogenic life style causing disease in animals and plants. Sclerotinia sclerotiorum is a pathogenic fungus, causing white mold, and infects a wide variety of crops such as soybean, green beans, sunflowers or peanuts. Phytophtora, belonging to the class of oomycetes, are plant pathogens infecting a wide variety of garden plants but also economical important food crops such as potato, fruit trees or soybean. Potato blight is caused by Phytophtora infestans which resulted in the Great Irish Famine between 1845-1849. Other Phytophtora species such as Phytophtora sojae or Phytophtora fragriae causes soybean or strawberry root rot. Disease management is difficult and often not successful. Infected plants and ideally soils have to be destroyed, tools in contact with the infected plants have to be cleaned and disinfected. Although some fungicides can act as a protectant, effective diseases control using chemicals is inefficient, resulting in the identification of resistant plants which are used in plant breeding programmes. However, long-term control using the above methods has shown to be inefficient.

This disclosure relates to inhibitors of LMPO expression in eukaryotic microbial species, in particular fungal and oomycetes pathogens of plants and fish. Typically, the agents comprise anti-sense RNAs, for example inhibitory RNAs such as siRNA.

Statements of the Invention

According to an aspect of the invention there is provide an inhibitory agent comprising an inhibitory RNA wherein said inhibitory RNA comprises an antisense nucleotide sequence complementary to a sense RNA molecule that encodes a lytic polysaccharide monooxygenase.

In a preferred embodiment of the invention said agent comprises an inhibitory RNA complementary to a sense RNA molecule encoding a fungal lytic polysaccharide monooxygenase.

In an alternative embodiment of the invention said agent comprises an inhibitory RNA complementary to a sense RNA molecule encoding an oomycete lytic polysaccharide monooxygenase.

In a preferred embodiment of the invention said agent comprises an inhibitory RNA that is complementary to a part of a sense nucleic acid molecule selected from the group consisting of: i) a nucleic acid molecule comprising a nucleotide sequence set forth in SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43 or 44; ii) a nucleic acid molecule that is at least 75% identical to the nucleotide sequence set forth in SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43 or 44 and that encodes a polypeptide with lytic polysaccharide monooxygenase activity.

Preferably, said agent comprises an inhibitory RNA that is complementary to a part of a sense nucleic acid molecule comprising a nucleotide sequence that is at least 80%, 85%, 90% or 95% identical to the nucleotide se, quence set forth in SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43 or 44 wherein said nucleotide sequence encodes a polypeptide with lytic polysaccharide monooxygenase activity.

In a preferred embodiment of the invention said agent comprises an inhibitory RNA that is complementary to a part of a sense nucleic acid molecule selected from the group consisting of: i) a nucleic acid molecule comprising a nucleotide sequence set forth in SEQ ID NO: 55, 56, 58, 59, 61 , 62, 64, 65, 67, 68, 70, 71 , 73, 74,

76, 77, 79, 80, 82, 83, 85, 86, 88, 89, 91 or 92; ii) a nucleic acid molecule that is at least 75% identical to the nucleotide sequence set forth in SEQ ID NO: 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65,66, 67and that encodes a polypeptide with lytic polysaccharide monooxygenase activity.

Preferably, said agent comprises an inhibitory RNA that is complementary to a part of a sense nucleic acid molecule comprising a nucleotide sequence that is at least 80%, 85%, 90% or 95% identical to the nucleotide sequence set forth in SEQ ID NO: 55, 56, 57, 58,

59, 60, 61 , 62, 63, 64, 65, 66 or 67 wherein said nucleotide sequence encodes a polypeptide with lytic polysaccharide monooxygenase activity.

In a preferred embodiment of the invention said agent comprises an inhibitory RNA that is complementary to a part of a sense nucleic acid molecule selected from the group consisting of: i) a nucleic acid molecule comprising a nucleotide sequence set forth in SEQ ID NO: 68, 69, 70, 71 , 72, 73, 74. 75. 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 ii) a nucleic acid molecule that is at least 50% identical to the nucleotide sequence set forth in SEQ ID NO: 68, 69, 70, 71 , 72, 73, 74. 75. 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 and that encodes a polypeptide with lytic polysaccharide monooxygenase activity.

Preferably, said agent comprises an inhibitory RNA that is complementary to a part of a sense nucleic acid molecule comprising a nucleotide sequence that is at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the nucleotide sequence set forth in

SEQ ID N068, 69, 70, 71 , 72, 73, 74. 75. 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102; wherein said nucleotide sequence encodes a polypeptide with lytic polysaccharide monooxygenase activity

In a further preferred embodiment of the invention said agent comprises an inhibitory RNA that is complementary to a part of a sense nucleic acid molecule selected from the group consisting of:

i) a nucleic acid molecule comprising a nucleotide sequence set forth in SEQ ID NO:103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113 or 114;

ii) a nucleic acid molecule that is at least 50% identical to the nucleotide sequence set forth in SEQ ID NO:103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113 or 114; and that encodes a polypeptide with lytic polysaccharide monooxygenase activity.

Preferably, said agent comprises an inhibitory RNA that is complementary to a part of a sense nucleic acid molecule comprising a nucleotide sequence that is at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the nucleotide sequence set forth in SEQ ID NO103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113 or 114 ; wherein said nucleotide sequence encodes a polypeptide with lytic polysaccharide monooxygenase activity.

In a further preferred embodiment of the invention said agent comprises an inhibitory RNA that is complementary to a part of a sense nucleic acid molecule selected from the group consisting of:

i) a nucleic acid molecule comprising a nucleotide sequence set forth in SEQ ID NO: 115, 116, 117, 118, 119 or 120; ii) a nucleic acid molecule that is at least 50% identical to the nucleotide sequence set forth in SEQ ID NO: 115, 116, 117, 118, 119 or 120and that encodes a polypeptide with lytic polysaccharide monooxygenase activity.

Preferably, said agent comprises an inhibitory RNA that is complementary to a part of a sense nucleic acid molecule comprising a nucleotide sequence that is at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the nucleotide sequence set forth in SEQ ID NO 115, 116, 117, 118, 119 or 120; wherein said nucleotide sequence encodes a polypeptide with lytic polysaccharide monooxygenase activity.

Although siRNAs should be designed downstream of the start codon of the mRNA, siRNA molecules can also be designed to the 3’ and 5’ untranslated regions (UTRs).

In a preferred embodiment of the invention said inhibitory RNA that is complementary to said part of said sense nucleic acid molecule comprises or consists of between 19 and 29 nucleotides or more preferably about 21 nucleotides.

In a preferred embodiment of the invention there is provided a composition comprising an agent according to the invention wherein said composition is adapted for spray application of said agent.

According to an aspect of the invention there is provided a composition comprising an agent according to the invention.

In a preferred embodiment of the invention said composition is adapted for spray application of said agent.

In a further preferred embodiment said composition comprises also a compound or a combination of compounds selected from the group consiting of:dimethyl-[3- (propoxycarbonylamino)propyl]azanium;chloride,2,6-dichioro-N-[[3-chioro-5

(trifiuoromethyl)pyridin-2-yl]mefbyl]benzamide, calcium chloride or calcium nitrate.

According to a further aspect of the invention there is provided a nucleic acid molecule comprising a transcription cassette wherein said cassette includes a nucleotide sequence of an inhibitory RNA according to the invention wherein said cassette is adapted for expression by provision of at least one promoter operably linked to said nucleotide sequence such that both sense and antisense molecules are transcribed from said cassette thereby expressing said inhibitory RNA. In a preferred embodiment of the invention said transcription cassette is part of an expression vector suitable for transformation of a plant.

A vector including nucleic acid according to the invention need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the gene.

Preferably the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, (e.g. bacterial), or plant cell. The vector may be a bi-functional expression vector which functions in multiple hosts. In the case of genomic DNA this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.

By "promoter" is meant a nucleotide sequence upstream from the transcriptional initiation site and which contains all the regulatory regions required for transcription. Suitable promoters include constitutive, tissue-specific, inducible, developmental or other promoters for expression in plant cells comprised in plants depending on design. Such promoters include viral, fungal, bacterial, animal and plant-derived promoters capable of functioning in plant cells.

Constitutive promoters include, for example CaMV 35S promoter (Odell et al. (1985) Nature 313, 9810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christian et al. (1989) Plant Mol. Biol. 18 (675-689); pEMU (Last et al. (1991) Theor Appl. Genet. 81 : 581-588); MAS (Velten et al. (1984) EMBO J. 3. 2723-2730); ALS promoter (U.S. Application No. 08/409,297), and the like. Other constitutive promoters include those in U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121 ; 5,569,597; 5,466,785; 5,399,680, 5,268,463; and 5,608,142.

Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induced gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize ln2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1 a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 and McNellis et al. (1998) Plant J. 14(2): 247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227: 229-237, and US Patent Nos. 5,814,618 and 5,789,156, herein incorporated by reference.

Where enhanced expression in particular tissues is desired, tissue-specific promoters can be utilised. Tissue-specific promoters include those described by Yamamoto et al. (1997) Plant J. 12(2): 255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7): 792-803; Hansen et al. (1997) Mol. Gen. Genet. 254(3): 337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3): 1331-1341 ; Van Camp et al. (1996) Plant Physiol. 112(2): 525-535; Canevascni et al. (1996) Plant Physiol. 112(2): 513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5): 773-778; Lam (1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al. (1993) Plant Mol. Biol. 23(6): 1129-1138; Mutsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90 (20): 9586-9590; and Guevara-Garcia et al (1993) Plant J. 4(3): 495-50.

"Operably linked" means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter. In a preferred aspect, the promoter is an inducible promoter or a developmental^ regulated promoter.

Particular of interest in the present context are nucleic acid constructs which operate as plant vectors. Specific procedures and vectors previously used with wide success upon plants are described by Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Cray RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148. Suitable vectors may include plant viral-derived vectors (see e.g. EP-A-194809).

If desired, selectable genetic markers may be included in the construct, such as those that confer selectable phenotypes such as resistance to antibodies or herbicides (e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate).

According to an aspect of the invention there is provided a plant transformed with the transcription cassette or expression vector according to the invention.

According to a further aspect of the invention there is provided the use an inhibitory agent comprising an inhibitory RNA wherein said inhibitory RNA comprises an antisense nucleotide sequence complementary to an RNA molecule that encodes a lytic polysaccharide monooxygenase in the control of eukaryotic microbial pathogens.

In a preferred embodiment of the invention said eukaryotic microbial pathogen is a fungal pathogen.

In a preferred embodiment of the invention said fungal pathogen is a plant fungal pathogen.

In a preferred embodiment of the invention said fungal pathogen is Botrytis.

In a further preferred embodiment of the invention said fungal pathogen is Botrytis cinerea.

In an alternative preferred embodiment of the invention said eukaryotic microbial pathogen is an oomycete pathogen. Preferably a plant oomycete pathogen.

In a preferred embdiment said plant oomycete pathogen s Phtythophora.

According to a further aspect of the invention there is provided an agent according to the invention adapted for spray application.

In a preferred embodiment of the invention said adaptation is for application as an areosol.

According to further aspect of the invention there is provided a method for the control of eukaryotic pathogenic microbial species comprising the steps: i) formulating a composition according to the invention; and ii) spray application of the composition to a plant or part of a plant to be treated.

It will be apparent that the compositions according to the invention may be applied prohylatically to a plant crop or as a treatment. Similarly it will be apparent to the skilled person that the compositions according to the invention may be applied post-harvest to, for example, fruit obtained from a plant to treat or prevent infection of the fruit. Moreover, said composition can be applied to plant growth substrates such as soil, eat or vermiculite or to hydropnic cultures.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of the words, for example“comprising” and“comprises”, means “including but not limited to”, and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

An embodiment of the invention will now be described by example only and with reference to the following figures:

Figure 1 (A) Dissected gut of T. domestica. The crop represents the largest portion of the foregut and the organ where food particles and digestive enzymes accumulate. (B) Microscopic image of Avicel® PH-101. Average particle size is ~ 50 pm. (C) Microscopic image of food pellet collected from the crop of T. domestica fed on Avicel® PH-101. Particle size is greatly reduced to ~ 5 pm. (D) HAPEC analysis of soluble extract isolated from the crop of T. domestica grown on Avicel® PH-101. One dominant peak corresponding to glucose is clearly visible, plus minor peaks for gluconic acid and cellobionic acid. Identity of the peaks was determined via analysis of commercial standards. (E) Pie chart summary of the CAZY enzymes identified in the crop of T. domestica fed with microcrystalline cellulose. Total protein obtained from pooled samples was subjected to trypsin hydrolysis and the peptides were searched against the transcriptome of Thermobia using Mascot. Molar percentage for each sequence was determined from emPAI scores and annotation of carbohydrate-active enzymes was performed with the online tools CAZYmes Analysis Toolkit (CAT) and dbCAN. Abundance values for the various families are: GH9 24.6%, LPMO 20.2%, GH18 13.2%, GH30 12.3%, GH1 8.4%, GH13 4.7%, GH31 4.0%, GH38 3.6%, GH35 2.1%, CE4 1.3%, GH2 1.1 %, GH20 1.0%, GH16 0.8%, AA3 0.7%, GH5 0.7%, GH27 0.6%, CE6 0.4%, GH65 0.3. (F) RT-PCR carried out with cDNA template generated from equal amount of RNA extracted from pooled samples of salivary glands, crop and midgut of adult T. domestica grown on microcrystalline cellulose. Oligonucleotide primers were designed for LPMO sequences GASN01405718.1 (a), GASN01404332.1 (b) and GASN01404396.1 (c). Strong, specific amplification of the target sequences was obtained only for cDNA from the midgut. (G) Full gene structure of LPMO GASN01030700.1 , showing the presence of three exons and two introns. The sequence was amplified from genomic DNA extracted from the legs of several specimens, cloned into a plasmid and sequenced via primer walking.

Figure 2: MALDI-TOF MS spectrum of products obtained after incubation of 0.4 mg/ml microcrystalline cellulose (A) or b-chitin (B) with 2 mM LPMO GASN01405718.1 and 1 mM ascorbic acid for 24 hours, showing native and oxidised oligosaccharides. For both substrates the main peaks correspond to sodium (+16) and disodium (+38) adducts of C1- aldonic acids. Smaller peaks for the monosodiated lactone (-2) were also identified. All oxidised species are marked in red. For chitin, the products released seem to be predominantly even-numbered oligosaccharides, implying that the enzyme can attack the crystalline structure, as previously observed for other LPMOs (7). (C) HPAEC chromatograms showing soluble cello-oligosaccharides released from microcrystalline cellulose by reactions containing combinations of recombinant LPMO, commercial GH6 and gallic acid. The main product is cellobiose. (D) HPAEC chromatograms showing soluble chito-oligosaccharides released from b-chitin by reactions containing combinations of recombinant LPMO, commercial chitinase and gallic acid. The main product is chitopentaose. (E) Synergy experiment showing release of cellobiose from microcrystalline cellulose by a commercial GH6. The presence of the LMPO boosts the activity of the GH6 by 5-fold, which further increases to 10-fold by addition of 1 mM gallic acid. (F) Synergy experiment showing release of chitopentaose from b-chitin by a commercial chitinase. The LPMO boosts the activity of the chitinase by 60-fold, increasing to 100-fold in presence of 1 mM gallic acid. All boosting experiments were carried out over 3 h at 28 °C and products quantified by HPAEC. Error bars represent s.e. of triplicate measurements.

Figure 3: Agar plate assays with soluble protein extract from T. domestics^’ s crop. Activity assays were carried on agar plates containing 0.1 % substrate and stained with Congo Red. Each plate was divided in three sectors, spotted either with buffer control (top left), T. domestics crop extract (top right) and positive control (bottom). Clearance zones are visible on all substrates, Indicating degradation of the polysaccharide by the protein extract of Thermobia;

Figure 4: Activity of the soluble extract of Thermobia^'s crop on a panel of polysaccharides, determined with the dinitrosalicylic acid assay (DNS). 50 mI reactions were performed in triplicate in a 96-well plate at 28 °C at 320 rpm using 2.8 micrograms of protein and 2 mg mL¹ substrate in 50 mM sodium phosphate buffer pH 6. After 3 hours, reactions were stopped by adding 100 mI DNS reagent and heating for 10 min at 100 °C, then absorbance at 540 nm was measured with a microplate reader. Bars indicate means (error bars: standard deviations of three replicates).

Figure 5 Purification and Thermofluor analysis of 7dAA14A. (A) SDS-PAGE analysis of periplasmic extract of E. coli (P), flowthrough after passing the periplasmic extract into a 5 mL strep-tag affinity chromatography column (FT), fractions of the protein peak eluted with 2.5 mM desthiobiotin (A7, A9, A10). (B) Melting curves of the recombinant LMPO after strep-tag affinity chromatography (Apo-Enzyme, T_m 58.5 °C), after metal loading with 5- fold molar excess of CuSCL (T_m 64 °C), size-exclusion chromatography (T_m 64 °C) and demetallation with 10 mM EDTA (T_m 58.6 °C). Significant increase in thermal stability was observed upon addition of copper and was retained after gel filtration, indicating tight specific binding and protein-fold rearrangement. Such increase was completely reversed by the metal chelator EDTA;

Figure 6: HPAEC chromatograms of the analysed reaction products from Td AA14A (2 mM) on PASC (4 mg mL^-1) in presence of gallic acid (4 mM final concentration). Assays were performed at 28 °C for 24 h under constant agitation (600 rpm}. Native oligosaccharides with a degree of polymerisation (DP) of 3 (ceiiotriose), 4 (cellotetraose), 5 (celiopentaose) and 6 (ceiiohexaose) and oxidised oligosaccharides (marked with an asterisk, *) were the main visible products. The identity of the peaks was determined by comparison with commercial standards and C1 -oxidised oligosaccharides generated by glucose oxidase (see “Materials and Methods” for more details). Although MALDI-MS of analogous reactions showed that longer native and oxidised oligosaccharides were also released by the LPMO, they could not be identified via HPAEC and are not visible in these chromatograms.

Figure 7: Activation of purified Td AA14A by different electron donors using PASC as substrate. Oligosaccharides with a degree of polymerisation from 2 to 6 were released from 1 mg ml_ ¹ PASC by the purified LPMO during 24-hour experiments in presence of 1 mM reductant and quantified via HPAEC. ASC: ascorbic acid. HQ: hydroquinone. GAL: gallic acid. PYR: pyrogallol. CYS: cysteine. QA: quinic acid. CA: coumaric acid. FA: ferulic acid. All experiments were done in triplicate. All values shown here have been blanked against reactions containing the LPMO without any electron donors (error bars: standard deviations of three replicates).

Figure 8 HPAEC chromatograms and histograms of synergy experiments with LPMO and commercial glycoside hydrolases on PASC. 100 pL reactions containing 1 mg mL ¹ substrate, 2 mM LPMO, 1 mM gallic acid and different amounts of commercial glycoside hydrolases (0.8 milli Units GH6, 5.4 milli Units GH7, 10 micrograms GH9 and 4 milli Units GH1) were carried out at 28 °C for 3 h at 600 rpm (see“Materials and methods” for more details). The LPMO boosted the activity of GH6, GH7, GH9 and GH1 by 40, 5, 20 and 200- fold. The main products released were cellobiose (GH6, GH7), cellotetraose (GH9) and glucose (GH1). All peaks were quantified by analysis of commercial standards. The chromatograms of all boosting experiments were staggered in order to avoid overlapping of the same peak from different samples. Bars indicate means (error bars: standard deviations of three replicates).

MATERIALS AND METHODS

Reagents

2,5-dihydroxy benzoic acid, ascorbic acid, gallic acid, pyrogallol, hydroquinone, cysteine, quinic acid, p-coumaric acid, ferulic acid, CuS0₄, Trizma-Base, HCI, Na₂HP04, NaH₂P04, sucrose, glucose, ampicillin and chloramphenicol were purchased from Sigma. Avicel® PH-101 (microcrystalline cellulose) was purchased from Sigma and prepared by sonicating a suspension in 1.8 mM acetic acid with a Misonix sonicator, until particle size was reduced to a size comparable to the one found in the crop of Thermobia fed on Avicel. The substrate was then washed several times in pure water until the pH reached 5.

PASC was prepared as follows. 5 g of Avicel were moistened with water and treated with 150 mL ice cold 85% phosphoric acid, stirred on an icebath for 1 hour. Then 500 mL cold acetone was added while stirring. The swollen cellulose was filtered on a glass-filter funnel and washed 3 times with 100 mL ice cold acetone and subsequently twice with 500 mL water. PASC was then suspended in 500 mL water and blended to homogeneity.

Pure squid pen chitin was kindly donated by Dominique Gillet (MAHTANI CHITOSAN Pvt. Ltd., India). High purity pachyman, tamarind xyloglucan, barley b-glucan, lichenan (from Icelandic moss), mannan (borohydride reduced), pachyman, konjac glucomannan, carob galactomannan, larch arabinogalactan and wheat arabinoxylan were purchased from Megazyme. Locust bean gum, carboxymethyl-cellulose (CMC) and beechwood xylan were purchased from Sigma.

Rearing of T. domestica

Animals were obtained from an online pet shop and grown at 38 °C in plastic containers with holes on the lid for aeration. A small glass beaker with water was placed in each container to provide the appropriate moisture. Minerals were provided in the form of a multivitamin powder, proteins in the form of soy protein isolate. The sources of carbon were powdered wheat straw, Whatman filter paper 1 , Avicel or blended oats. After feeding for at least two weeks on these diets, animals were euthanized in ice and dissected under a stereo-microscope with sterile tools.

Shotgun proteomics

Crops from eight animals grown on a specific diet (wheat straw, filter paper, Avicel or oats) were dissected in 50 mM sodium phosphate buffer pH 7 and the content (food particles and enzymes) was collected, added with 1% SDS, beta-mercapto ethanol, DTT, boiled for ten minutes, centrifuged and the supernatant shortly run in a 10% polyacrylamide gel.

In-gel tryptic digestion was performed post reduction with DTE and S- carbamidomethylation with iodoacetamide. Resulting peptides were analysed by label free LC-MS/MS over a 125 min gradient using a Waters nanoAcquity UPLC interfaced to a Bruker maXis HD mass spectrometer as detailed in (29). Protein identification was performed by searching tandem mass spectra against a downloaded copy of the transcriptome of T. domestica (BioSample: SAMN02047119; Sample name: INSbttTSRAAPEI-29 - Thermobia domestica; SRA: SRS462938) using the Mascot search program. Matches were passed through Mascot percolator to achieve a false discovery rate of <1 % and further filtered to accept only peptides with expect scores of 0.05 or better. Molar percentages were calculated from Mascot emPAI values by expressing individual values as a percentage of the sum of all emPAI values in the sample (30). Proteins identified in the proteomics analysis were annotated via Blastx versus non-redundant NCBI databases. CAZy annotation was carried out using the CAZYmes Analysis Toolkit (CAT) on the BioEnergy Science Center website (http://mothra.ornl.gov/cgi-bin/cat/cat.cgi) and dbCAN (http://csbl.bmb.uqa.edu/dbCAN). A specific search was set up in Mascot using histidine methylation as a variable parameter, but only masses compatible with a non-methylated histidine a the N-terminus were found.

Phylogeny and classification of the new LPMOs

The new LPMO sequences identified in the transcriptome of Thermobia were searched via BlastP against NCBI non-redundant databases. A total of 191 AA14 sequences (169 sequences from NCBI plus 22 Thermobia sequences) were analysed for phylogeny. To avoid interference from the presence or absence of additional modules, the signal peptides and C-terminal extensions were removed. The resulting amino acid sequences corresponding to the catalytic domain were aligned using Muscle (31), operating with default parameters. A distance matrix was derived from the alignment using Blosum62 substitution parameters (32) and subsequently used to build a phylogenetic tree using the neighbor-joining method (33). The resulting tree was visualized using Dendroscope (34) and edited with the graphic tools Gimp and CorelDraw.

Cloning the full length gene of LPMO GASN01030700.1

Genomic DNA was extracted from the legs of ten animals using the DNeasy Blood and Tissue Kit (Qiagen). External primers designed for contig GASN01030700.1 were used to amplify the full gene (from start to stop codon) using genomic DNA as template and CloneAmp polymerase (Clontech) via nested PCR. The product, with estimated size of 4.5 kbp, was cloned via StrataClone Blunt PCR Cloning Kit (Stratagene). The gene structure was then determined via Sanger sequencing using internal primers. Intron/exon boundaries were identified by comparing the full gene sequence with the coding sequence from the cDNA.

RT-PCR of LPMO seguences

Salivary glands, crop and anterior midgut were dissected from ten animals grown on Avicel and the total RNA was extracted with the TRIzol® Reagent (Thermo Fisher Scientific). cDNA was generated with an oligodT primer using Superscript® II reverse transcriptase (Thermo Fisher Scientific) in 20 pL reactions containing 300 ng RNA. 0.3 pL of cDNA was used as template in 15 pL PCR reactions to amplify LPMO coding sequences using Phusion® High-Fidelity DNA Polymerase (New England Biolabs) and sequence- specific oligonucleotide primers. Cloning, heterologous expression and purification of recombinant protein Native sequences coding for LPMOs were cloned from cDNA generated from RNA extracted from Thermobia. Briefly, total RNA was extracted from one animal using the TRIzol® Reagent (Thermo Fisher Scientific) and cDNA was generated with an oligodT primer using Superscript® II reverse transcriptase (Thermo Fisher Scientific).

The coding sequences starting from the codon of the catalytic histidine were either cloned into an auxiliary plasmid via the StrataClone Blunt PCR Cloning Kit (Stratagene) or directly into a modified pET26b after the pelB leader sequence and C-terminally fused to a strep- tag.

The expression plasmid carrying the LPMO sequence was transformed into E. coli Rosetta 2 (DE3) pLysS (Novagen) via heat shock. A single colony was inoculated into LB medium plus 100 pg mL ¹ ampicillin and 34 pg mL ¹ chloramphenicol and grown overnight at 100 rpm at 30 °C. In the morning, 10 mL of this starter culture were used to inoculate 1 L of M9 minimal salts medium containing 1 % (w/v) glucose and the appropriate antibiotics. The cell culture was grown at 210 rpm at 37 °C until OD600 reached 0.7, then induced with 1 mM IPTG and left overnight at 20 °C. After protein expression, cells were harvested, re suspended in ice cold 50 mM Tris HCI pH 8 with 20% (w/v) sucrose and left in ice for 30 minutes before centrifugation. The supernatant was discarded and the pellet was re- suspended in ice cold 5 mM MgSCL plus 100 pM AEBSF protease inhibitor and left in ice for 30 minutes. After centrifugation, the supernatant was collected, filtered and the pH adjusted to 7.6 with 200 mM Na phosphate buffer. The periplasmic extract was then injected into a 5 mL StrepTrap HP column (Ge Healthcare) and, after washing with binding buffer, the protein was eluted with 2.5 mM desthiobiotin. Protein concentration was determined either via Bradford assay or via absorbance at 280 nm with a NanoDrop spectrophotometer (using molecular weight 22400 and extinction coefficient 40000 for the mature, strep-tagged protein). 5 fold excess copper was added as CuS0₄, then unbound copper and desthiobiotin were removed by passing the protein in a HiLoad™ 16/60 Superdex 75 gel filtration column (Ge Healthcare) equilibrated with 10 mM sodium phosphate buffer pH 7. The protein was then concentrated using Microsep™ Advance Centrifugal Devices (Pall Corporation).

Thermal shift assay (Thermofluor)

The Thermofluor assay was conducted on the purified protein with SYPRO® Orange Protein Gel Stain (Life Technologies) using an Mx3005P qPCR System (Agilent Technologies). The intensity of the fluorescence was measured at a temperature gradient of 25-95 °C and converted into a melting curve (fluorescence changes against temperature) to determine the melting temperature (T_m).

ESI-FTICR-MS analysis

Purified 7d(AA14)B was submitted for FTICR-MS analysis. The sample was split in two, half the material was reduced with (tris (2-carboxyethyl) phosphine), the other half was untreated. Both samples were alkylated with methanethiosulfonate to give methylthio- modified Cys residues at non disulfide bonded positions. FTICR-MS acquisition was performed using a Bruker solariX instrument as detailed in (35).

Activity assays

Activity of the crop extract on a panel of substrates was determined by reducing sugar assay. Briefly, crops were dissected in 20 mM sodium phosphate buffer pH 6 and the content fully resuspended by pipetting. After centrifugation, the soluble portion (supernatant) was filtered through 0.22 pm porous membranes, quantified with the Bradford reagent and used for assays. Briefly, the typical 50 pL reaction was carried out in 96-well plates in 50 mM sodium phosphate buffer pH 6 with 2.8 pg of protein and 2 mg ml_ ¹ substrate. All reactions, including controls, were performed in triplicate. The microplate was incubated at 28 °C shaking at 320 rpm for 3 hours, then 100 pL of DNS reagent were added to each reaction before heating at 100 °C for 5 min. Absorbance at 540 nm was measured with a micro-plate reader and nanomoles of reducing sugars released were determined based on absorbance obtained with glucose standards. The DNS reagent was prepared by mixing 0.75 g of dinitrosalycilic acid, 1.4 g NaOH, 21.6 g sodium potassium tartrate tetrahydrate, 0.53 ml_ phenol and 0.59 g sodium metabisulfite in 100 ml_ pure water.

Plate assays were carried out by spotting 10 pL of soluble crop extract (concentration 0.56 mg ml_ ¹) on 1.2% agar plates containing 0.1 % (w/v) substrate. After incubation at 28 °C for 16 hours, the plates were covered with Congo Red solution (0.1 % w/v Congo Red in 5 mM NaOH) for 30 min at room temperature, then washed with 1 M NaCI and visualized. Activity was indicated by clearance zones. A 1/100 dilution of Celluclast® (Novozymes) was used a positive control.

Typical reactions for LPMO characterization were carried out by mixing 1-4 mg ml_ ¹ substrate (PASC, Avicel or b-chitin) with purified 7cf(AA14)A or 7cf(AA14)B (2 uM), 1-4 mM electron donor, in a total volume of 100 pL in 2 ml_ plastic reaction tubes. All reactions analyzed via MALDI were carried out in 50 mM ammonium acetate buffer pH 6 and incubated at 28 °C shaking at 600 rpm and the supernatant used for analysis.

Reactions used for product quantification and boosting experiments were typically carried out in 50 mM sodium phosphate buffer pH 6 in triplicates of 100 pl_ each for 3 hours at 600 rpm at 28 °C. Each reaction contained 2 mM purified 7d(AA14)A, 1-4 mg ml_ ¹ substrate and 1 mM electron donor. Commercial GH6 (cat. number E-CBHIIM, Megazyme), GH7 (cat. number E-CELTR, Megazyme), GH9 (cat. number CZ03921 , ZNYTech), GH1 (cat. number E-BGOSAG, Megazyme) and chitinase (cat. number C6137-5UN, Sigma) were added to 100 mI_ reactions. After 3 hour incubation, 400 mI_ of ethanol were added to stop the reaction, spun down and 400 mI_ of supernatant were transferred to new plastic tubes, dried down and re-suspended in 80 mI_ of pure water, filtered and analyzed via HPAEC.

Standards of C1 -oxidised oligosaccharides were produced with commercial glucose oxidase. Briefly, 200 mI_ reactions containing 100 mM glucose oxidase from Aspergillus niger (Sigma) and 500 mM substrate (cellobiose, cellotriose, cellotetraose) were carried out in in 50 mM sodium phosphate buffer pH 6 for 24 h under constant agitation (600 rpm). The reactions were stopped by adding 800 mI_ ethanol, spun down and 800 mI_ of supernatant were collected, dried in vacuo and re-suspended in 160 mI_ pure water for analysis. For all substrates, negative control reactions without enzyme were also carried out.

Product analysis by HPLC

Oligosaccharides were analyzed from undiluted samples via HPAEC using a ICS- 3000 PAD system with an electrochemical gold electrode, a CarboPac PA20 3x150 mm analytical column and a CarboPac PA20 3x30 mm guard column (Dionex). Sample aliquots of 5 mI_ were injected and separated at a flow rate of 0.5 ml_ min ¹ at a constant temperature of 30 °C. After equilibration of the column with 50%-50% H₂O-O.2 M NaOH, a 30 min linear gradient was started from 0 to 20% with 0.5 M sodium acetate in 0.2 M NaOH and then kept constant for 20 minutes. The column was then washed with 0.2 M NaOH for 6 min and re-equilibrated for 4 min with 50%-50% H₂O-O.2 M NaOH before starting the next run. Integrated peak areas were compared to mono and oligo-saccharide calibration standards (glucose, cellobiose, cellotriose, cellotetraose, cellopentaose, cellohexaose, N- acetylglucosamine, chitobiose, chitotriose, chitotetraose, chitopentaose) obtained from Megazyme. Product analysis by mass spectrometry (MS)

1 mI_ of reaction supernatant was mixed with an equal volume of 20 mg ml_ ¹ 2,5- dihydroxybenzoic acid (DHB) in 50% acetonitrile, 0.1 % TFA on a SCOUT-MTP 384 target plate (Bruker). The spotted samples were then dried in a vacuum desiccator before being analyzed by mass spectrometry on an Ultraflex III matrix-assisted laser desorption ionization-time of flight/time of flight (MALDI/TOF-TOF) instrument (Bruker) as previously described (36).

Sample permethylation was carried out according to (37). Spotted samples were analyzed by MS using 2,5-DHB matrix with 0.1 % TFA on an AB-Sciex 4700 (for MALDI-TOF) and Ultraflex III MALDI/TOF-TOF instrument (Bruker). Data were collected using a 2kHz smartbeam-ll laser and acquired on reflector mode (mass range 300-3000 Da) for MS analysis and on LIFT-CID for MS/MS analysis using argon as collision gas. FlexControl and FlexAnalysis softwares were used for data acquisition and analysis. On average, about 10000 shots were used to obtain high-enough resolution. MS/MS fragmentation patterns were named according to (38).

Crystallization, X-ray data collection and structure determination of 7^~c/(AA14)A

Sitting drop crystallization screens were set up using copper-loaded Td(AA14)A at 10 mg mL ¹ using fomulatrix NT8 robotics. Initial crystal hits were obtained in the JCSG Core I and II screens (Qiagen), conditions F11 and H11 respectively. These crystals were subsequently optimized in further sitting-drop vapor diffusion experiments mixing 0.2 pL of the protein at 10 mg mL ¹ with 0.1 pL of crystallization solution - 0.1 M sodium citrate pH 5.5, 0.1 M LiCI, and 10 to 25% w/v polyethylene glycol 6000 (PEG-6000). All screens were performed at 20 °C.

Crystals were cryo-protected by soaking in mother liquor supplemented with 20% ethylene glycol before being plunged in liquid nitrogen. Data were then collected at the ESRF, MASIF-1 beamline at a fixed wavelength of 0.966 A. Ten datasets were collected without manual intervention, five of which were collected using the MXPressE_SAD protocol to allow attempts at experimental phasing using the weak anomalous signal that would be obtained from the copper at this wavelength, and five datasets were collected using the MXPressE protocol to provide the best possible native data. All datasets were indexed using XDS (39). Individual datasets were processed using CCP4 (40) but these did not contain sufficient anomalous signal to allow structure determination. All five datasets collected using the MXPressE_SAD method were, therefore, combined and scaled using BLEND (41) to 2 A resolution. The structure was then successfully determined from the copper anomalous signal using SHELX (42). The initial structure was rebuilt using BUCCANEER (43), and this model was then refined against the best native dataset at 1.1 A resolution. Subsequent rounds of manual rebuilding and refinement were performed in COOT (44) and REFMAC5 (45), respectively. The quality of the model was monitored throughout rebuilding and refinement using MolProbity (46), with the final model containing a single Ramachandran outlier (Ser33) and 96.9% of residues in the favored region of the Ramachandran plot. Data processing and structure refinement statistics are shown in Table S5.

The structure and accompanying structure factors have been deposited in the Protein Data Bank with accession code 5MSZ.

ConSurf Analysis

For ConSurf analysis we generated an alignment using 193 publicly available sequences defined as being in this LPMO family in CAZy, using MUSCLE (31). The 21 sequences identified in the current study were then added to the alignment using MAFFT (47), giving a final alignment containing 214 sequences from the same family. This alignment was then uploaded to the ConSurf server for analysis (48), ensuring that only LPMOs in the same family were analyzed. The ConSurf scores were visualized on the protein surface using PyMol.

Electron paramagnetic resonance analysis (EPR)

Continuous wave X-band frozen solution EPR spectra of single sample of 0.2 mM solutions of Cu(ll)-Td(AA14)A (in 10% v/v glycerol) at pH 7.0 (50 mM sodium phosphate buffer) and 160 K were acquired on a Bruker EMX spectrometer operating at -9.30 GHz, with a modulation amplitude of 4 G and microwave power of 10.02 mW. Spectral simulations were carried out using EasySpin 5.0.3 (49) integrated into MATLAB R2014a software (50) on a desktop PC. Simulation parameters are given in Table 1. g_z and |A_Z| values were determined accurately from the three absorptions at low field. It was assumed that g and A tensors were axially coincident. Accurate determination of the gx, gy, |Ax| and |Ay| was not possible due to the presence of two species and the second order nature of the perpendicular region, although it was noted that satisfactory simulation could only be achieved with the particular set of g values reported in Table 1. Raw EPR data are available on request through the Research Data York (DOI: 10.15124/bd09e86b-9d92- 4802-9337-18b138e7abb7). Table 1.

EPR spin Hamiltonian parameters from simulations of cw X band spectra for 7d(AA14)A in the presence of 10% glycerol in 50 mM Na phosphate buffer pH 7 at 160 K. Best fit obtained with 15% of species 2. Parallel values are determined accurately. Accurate determination of perpendicular values and superhyperfine coupling constants was not possible due to second order nature of the spectrum in this region.

Species 1 Species 2

9^c 2.02 2Ό4

g values g_y 2.11 2.08

9z 2.283 2.258

Ax 15 45

Ac_u (MHz) A_y 65 55

A_z 407 512

SHF A_N (isotropic) (MHz) 23, 31 , 35 31 , 35

Acu strains (MHz) 190, 65, 130 57, 65, 120

Line widths (Gaussian, Lorentzian) 0.2, 0.2 0.2, 0.2

Methods for Testing Agents comprising siRNA directed to LMPOs

Testing ability of silencing constructs targeting LPMOs from Hyaloperonospora arabidopsidis and Botrytis cinerea and Phytophthora infestans to limit disease progression.

Testing can be carried out using stably transformed plants (host-induced silencing) or by treatment of plants with RNA constructs.

Generation of transformed Arabidopsis plants expressing LPMO silencing constructs: PCR amplified sequences (400-500 bp) from lytic polysaccharide monooxygenase genes in Hyaloperonospora arabidopsidis and Botrytis cinerea are inserted into the Gateway silencing vector PB7GWIWG2(I) (Karimi, M., Inze, D., Depicker, A., Gateway vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci. 2002 May;7(5): 193-195) to generate constitutively expressed silencing constructs. Arabidopsis thaliana Col-0 plants are transformed using the floral dip method (Clough and Bent, Plant Journal 1998 Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. (6):735-43) and transformed plants selected using soil-drenching with 150 mg/L phosphinotricin.

Transformed plants are assessed for expression of the silencing construct using quantitative PCR and compared to wildtype untransformed siblings for susceptibility to H. arabidopsidis and B. cinerea. Treatment of plants with double stranded RNA

DNA template (approximately 700 bp) for double stranded RNA synthesis is generated by polymerase chain reaction from genomic DNA or a cloned sequence. Double stranded RNA is synthesised via in vitro transcription using primers containing the T7 RNA polymerase promoter sequence (Beckert B., Masquida B. (2011) Synthesis of RNA by In Vitro Transcription. In: Nielsen H. (eds) RNA. Methods in Molecular Biology (Methods and Protocols), vol 703. Humana Press). Double stranded RNA is sprayed onto the surface of plant leaves prior to infection assays.

B. cinerea disease assessment:

Arabidopsis plants are grown and susceptibility to B. cinerea assessed as described in Windram et al. (2012) Arabidopsis defense against Botrytis cinerea : chronology and regulation deciphered by high-resolution temporal transcriptomic analysis. Plant Cell 24(9): 3530-3557. Briefly, Arabidopsis plants are grown under a 16:8 hr light:dark cycle at 23°C, 60% humidity and light intensity of 100 pmol photons. nr².s ¹. Arabidopsis seed were stratified for three days in 0.1% agarose at 4°C before sowing onto Arabidopsis soil mix (Scotts Levingtons F2s compost:sand:fine grade vermiculite in a ratio of 6:1 :1).

B. cinerea strain pepper (Denby, K.J., Kumar, P., and Kliebenstein, D.J. (2004). Identification of Botrytis cinerea susceptibility loci in Arabidopsis thaliana. Plant J. 38: 473- 486) is sub-cultured on sterile tinned apricot halves in petri dishes two weeks prior to use of the spores. Sub-cultures are incubated in the dark at 25°C. Spore inoculums are prepared by harvesting spores in water, filtration through glass wool to remove hyphae and suspension in half strength sterile grape juice to a concentration of 1 x 10⁵ spores/mL. For assaying susceptibility of plant lines, leaves from 4-week old plants are detached and and placed on a bed of 0.8% agar in propagator trays. A single 10 pL droplet of B. cinerea inoculum or sterile half strength grape juice (mock control) is placed in the centre of each leaf. Trays are covered with lids and kept under the same conditions as for plant growth, except the relative humidity is raised to 90%. Lesion perimeters at time points after inoculation are determined from photographs using the image analysis software ImageJ (http://rsb.info.nih.gov/ii/). Mean lesion perimeters of at least 30 leaves will be assessed.

H. Arabidopsis disease assessment

Assessment of susceptibility to H. arabidopsidis in transformed Arabidopsis plants expressing LPMO silencing constructs and wildtype untransformed siblings is carried out as described in Tome D.F.A., Steinbrenner J., Beynon J.L. (2014) A Growth Quantification Assay for Hya!operonospora arabidopsidis isolates in Arabidopsis thaiiana. in: Birch P., Jones J., Bos J. (eds) Plant-Pathogen interactions. Methods in Molecular Biology (Methods and Protocols), vol 1127. Humana Press, Totowa, NJ Arabidopsis seed is sown at a density of approximately 25 seedlings per module (of a P40 seed tray) and grown for 14 days under 12 h light, 20°C, 60% humidity and light intensity of 100 pmol photons. nr². s ¹ Spores of H. arabidopsidis isolate Emoy2 are collected from previously infected seedlings by placing seedlings with 50 mL water and shaking vigorously. The spore suspension is filtered through miracloth and adjusted with water to a concentration of 3 x 10⁴ spores/mL. Spore suspension is sprayed evenly onto seedlings using 15 mL of inoculum per P40 seed tray of seedlings. Propagator lids are replaced and placed in a growth cabinet with 10 h light, 18°C, and light at 100 pmol photons. nr².s ¹· Four days post infection, the infection is quantified by counting total sporangiophores per individual seedling using a dissecting microscope. Fifteen seedlings per module are counted with at least 3 modules per Arabidopsis line.

Phytophthora infestans disease assessment:

Assessment of Nicotiana benthamiana to P. infestans is assessed as outlined in McLellan et al. (2013, An RxLR Effector from Phytophthora infestans Prevents Re-localisation of Two Plant NAG Transcription Factors from the Endoplasmic Reticulum to the Nucleus. PLoS Pathog 9(10): e 1003670. https://doi.orq/10.1371/iournal.ppat.1003670). P. infestans is cultured on rye agar and sporangia collected. Suspensions of ~1.5 x 10³ sporangia mL are inoculated as 10 P L droplets onto detached leaves and leaves incubated at high humidity. The number of sporulating lesions as well as number of sporangia produced is counted days after infection. Protocol for infecting detached leaves from Lettuce ILactuca sativa) with i)

Sclerotinia sclerotiomm

Detached leaf infection procedure Settings for Sanyo 970 controlled environment chamber:

Light - 16 hours (0500 - 1700)

Temperature - 22°C

Humidity - 95%

CO2 - 350ppm

Set up of Agar trays

1. Add 6.4g of agar-agar to 800ml of purite water (0.8% agar) in a 1 L bottle. Swirl to thoroughly mix

2. Autoclave and leave to cool in 40-60°C oven (media that is too hot will melt the salad trays!)

3. Pour half of each bottle (400ml) into each salad tray.

4. Leave salad tray in a class I flow hood to dry.

5. Close the lid once dried and cooled.

Lettuce leaf collection & experimental set up

1. Label small grip seal polythene bags with line number

2. Identify leaf 3¹ for each lettuce plant. Ignoring the cotyledons count the true

leaves from the outside in. The leaf should be fully expanded and all leaves should be of a similar developmental stage.

3. Detach leaf 3 from the plant using scissors and place in a sealed polythene bag.

Place these in a cool box.

4. Leaves and trays should be transported to experimental location in the main building. 5. Prior to arranging the leaves on the agar sort the leaves into numerical order on the bench to make following the randomisation easier

6. Place laminated tray label and scale on the agar in the top left hand corner of tray

7. Starting in position 1 , the top left hand corner of the tray, lay the leaves out in order of the randomisation working across the tray from left to right and top to bottom.

8. Arrange the leaves to fit ensuring the cut end of the leaf is placed just into the agar

9. If a plant line is missing place a green paper square on the agar where it should be in the randomisation

10. Close the lid of the salad tray and ensure the rim is sealed all the way around.

Sclerotinia inoculum

1. Cut a ¼ of a circular filter paper containing spores of isolate and re-suspend in 5 ml_ sterile distilled water (SDW) in a plastic bijou bottle.

2. Shake it gently to produce a visibly cloudy suspension

3. Place a folded strip of miracloth into the bottle and filter into a clean bijou bottle

4. Count spore concentration using a haemocytometer and adjust to

500,000spore/ml in final concentration of 50% potato dextrose broth and 1 % guar gum.

Infection of detached lettuce leaves

Holding tip just above leaf surface pipette two spots each of 5 ul either side of the midvein. Gently shake the inoculum in between trays to re-suspend spores. Put lids on propagator trays and place in growth room.

Assessment of disease severity

Lesion symptoms may be just about visible at 24 hours post infection (hpi), however they will be difficult to quantify. First true lesions will be quantifiable at 48 hpi. Generally, 64hpi provides the optimum time to assess infection. 72hpi shows large lesions but may overrun the leaf edge. Photographs should be taken at 48, 64 & 72 hours post infection. Lesion size is assessed using ImageJ.

The Protocol for infecting leaves from plants such as potato with P. infestans are described in Whisson et al. , 2007, Nature Letters, Vol 450, A translocation signal for delivery of oomycete effector proteins into host plant cells. Example 1

To investigate the digestive enzymes produced by T. domestica, we grew batches of individuals on different carbon sources and isolated the content of the crop, which represents the largest organ of the foregut (Fig. 1A). Microscopic analysis of the crop content from animals grown on microcrystalline cellulose (Avicel) revealed that the particle size of cellulose was markedly reduced (Fig. 1 B and Fig. 1C). HPAEC analysis of the fluids of the crop from animals grown on Avicel showed a dominant peak corresponding to glucose, indicating that crystalline cellulose had been broken down to its monomeric unit (Fig. 1 D). Agar plate and in vitro activity assays carried out with the soluble proteins extracted from the crop revealed the ability to breakdown a wide range of complex polysaccharides normally found in plant biomass, including glucans, mannans and xylans (Fig. 3 and 4), suggesting the presence of a complex enzymatic cocktail.

To identify the enzymes responsible for the breakdown of polysaccharides, we performed shotgun proteomics of the crop content from T. domestica that had been fed on either oat flour, pulverized wheat straw, filter paper, or Avicel as the main carbon source. Tandem mass spectra were searched with Mascot software against the publicly available T. domestica transcriptome published by the 1 K Insect Transcriptome Evolution (1 KITE) initiative (NCBI BioProject PRJNA219608). Identified proteins were ranked by emPAI derived molar percentage abundances (14).

Our analysis revealed that the crop proteome is dominated by recognizable CAZymes that make up around 50% of the total protein content, with proteases and structural proteins also abundant. Interrogation of public databases showed that more than 90% of the proteins found in the gut of T. domestica have best matches with genomic sequences from insects and crustaceans, with the exception of a small number of putative bacterial glycoside hydrolases belonging to family 30 (GH30s) from Bacillus species , suggesting only minor microbial contributions to the digestive proteome.

Amongst the identified proteins was a group of unknown function that showed increasing abundance in animals fed on cellulose-enriched diets, representing 7%, 8%, 10% and 20% of the total CAZymes for animals grown on oats, paper, wheat straw and microcrystalline cellulose, respectively (Fig. 1 E). Our proteomics data indicated the presence of 21 such proteins with corresponding ESTs in the T. domestica transcriptome. Sequence analysis revealed that these proteins share very distant similarity to known LPMOs, limited to the conserved N-terminal histidine brace that coordinates the active site copper (13). Sequence interrogation of public databases and phylogenetic analysis further showed that the newly identified proteins belong to a distinct family of LPMOs that occurs in a range of invertebrate lineages including insects, crustaceans and mollusks (Fig. 2). Related proteins are also present in a range of algal species and in oomycetes, none of which have previously been shown to possess LPMOs. The significant similarity between the T. domestica sequences and their orthologs, and the lack of significant similarity with previously established LPMO families (AA9, AA10, AA11 and AA13) allow us to define a new LPMO family, which will appear as AA14 in the CAZy database.

While sequences in T. domestica only correspond to the LPMO catalytic domain, about a third of the members of the family identified in other species harbor a C-terminal extension. Most of these C-terminal extensions can be assigned to various carbohydrate-binding domain (CBM) families based on amino acid sequence relatedness (for classification of CBMs see CAZy database). The fusion of AA14 members to CBM1 (cellulose-specific) and CBM 14 (chitin-specific) domains suggests that this new LPMO family could potentially target both cellulose and chitin (15). In addition, some of the identified AA14 LPMOs are fused to GH18 (Chlorophyta, Bacillariophyceae and tunicates) or GH19 (Oomycota, Haptophyta) domains, both classified as chitinases .

Out of 22 full length LPMO catalytic domain encoding sequences identified in the transcriptome of T. domestica, peptides representing 21 were detected in significant amounts in the gut proteome. Such LPMO diversification within a single organism has been previously observed in fungi and might indicate isoform-specific preference towards different substrates, electron donors, pH and temperatures (16).

Protein sequence analysis revealed that all LPMOs from T. domestica carry a signal peptide that, once removed, allows the exposure of the conserved N-terminal catalytic histidine of the mature, secreted protein. Ten cysteine residues (potentially forming stabilizing disulfide bridges in the proteins) were found to be conserved both in the T. domestica LPMOs and in the best BlastP matches found in crustaceans, mollusks, insects and spiders. Fungal LPMOs possess an N-terminal methylated histidine but this was not observed in proteomic analyses of the LPMOs from T. domestica. To confirm the presence of the newly identified LPMO sequences in the transcriptome of T. domestica, we amplified, cloned and sequenced a number of putative LPMO cDNAs generated from polyadenylated RNA. Reverse transcription PCR (RT-PCR) performed with cDNA extracted from salivary glands, crop and midgut showed that the LPMO genes were most highly expressed in the midgut (Fig. 1 F). In order to avoid contamination by DNA from digestive tract microbes, we extracted genomic DNA from the legs of several T. domestica and used this as a template to amplify the gene of one LPMO. The amplified sequence, measuring 4.5 kbp, was cloned and sequenced, revealing the presence of three exons and two introns (Fig. 1G), supporting the endogenous origin of these enzymes.

Example 2

Coding sequences representing two putative LPMOs from T. domestica (contigs GASN01405718.1 and GASN01404332.1, henceforth termed Td(AA14)A and Td(AA14)B, respectively) were cloned, expressed in Escherichia coli, and the recombinant proteins were purified (Fig. 5). Because gene expression was carried out using a minimal medium devoid of metals, the purified LPMOs were not bound to copper. Thermal shift analysis (Thermofluor) of purified apo-Td(AA14)A indicated a melting temperature (T_m) of 58.5 °C, which increased to 64 °C upon addition of excess copper and was retained after size exclusion chromatography (Fig. 5). Stripping of copper with 10 mM EDTA lowered the T_m back to 58.6 °C. Similar thermal shifts were also obtained with purified Td(AA14)B (data not shown). These results indicate that the apo-enzyme folds correctly in the periplasm of E. coli and that subsequent addition of copper increases the T_m and protein stability, as observed with other LPMOs (17).

Activity assays were initially carried out with purified Td(AA14)A and Td(AA14)B on polysaccharides (1-4 mg mL ¹) in the presence of ascorbic acid, which has commonly been used as an electron donor to test LPMO activities. LPMO activity assays were carried out using Avicel, phosphoric acid swollen cellulose (PASO) and squid pen chitin (b-chitin). Samples were analyzed via MALDI-TOF MS. Peak masses of the reaction products were compared to previously published data (6, 7, 8) and are compatible with a predominant C1 -oxidation pattern and generation of C1-aldonic acids on all three substrates by both Td(AA14)A and Td(AA14)B (Fig. 2A and 2B).

We focused our attention on Td(AA14)A and confirmed C1-oxidation by MALDI-TOF MS and MS/MS analysis of the permethylated cellulose cleavage products. The supernatant from reactions with PASO and microcrystalline cellulose was also analyzed by HPAEC, confirming production of both native and oxidized oligosaccharides (Fig. 6). It has been widely reported that LPMOs can accept electrons from several small molecular reductants, including phenolics derived from lignin (18). We therefore screened a panel of compounds for their ability to drive the activity of 7d(AA14)A on PASC and identified gallic acid as the most effective reductant (Fig. 7).

LPMOs can enhance the saccharification of recalcitrant polysaccharides by glycoside hydrolases (6, 9, 10). We carried out synergy experiments with commercially relevant cellulases and chitinases and analyzed the released products by HPAEC. Reactions containing either 7d(AA14)A or the glycoside hydrolase alone released only small amounts of oligosaccharides from cellulose or chitin, while co-incubation reactions containing both enzymes dramatically increased the yield. Such boosting effects were further enhanced by addition of gallic acid, reaching a maximum 40-fold, 5-fold, 20-fold and 200-fold improvement with GH6, GH7, GH9 and GH1 enzymes respectively when using PASC as substrate. The LPMO also boosted the activity of a GH6 on microcrystalline cellulose more than 25-fold and the activity of a chitinase on b-chitin 150-fold (Fig. 3C, 3D, 3E, 3F, 8). To our knowledge, this is the first example of a single LPMO boosting the activity of glycoside hydrolases on both cellulose and chitin.

Example 3

To elucidate the structural basis of the unusual substrate promiscuity of Td(AA14)A, we determined its crystal structure to 1.1 A resolution by single-wavelength anomalous diffraction (SAD) phasing using the active site copper center as the anomalous scatterer . The model shows that the core protein fold is highly reminiscent of other LPMOs consisting of a central b-sandwich fold decorated with diverse loops that link the strands together and is stabilized by five disulfide bonds. Five disulfide bonds were also independently identified in Td(AA14)B via ESI-FTICR-MS analysis and might therefore be crucial for correct folding of all Thermobia^'s LPMOs. The structure of Td(AA14)A is also characterized by the ubiquitous LPMO histidine brace formed by His1 and His91. Since the enzyme was heterologously produced in E. coli, His1 was not methylated and therefore represented the state of the native protein from T. domestica.

The Dali server was used to compare the structure more widely with other structures in the Protein Data Bank (PDB) (19). This analysis revealed that Td(AA14)A most closely resembles bacterial AA10 LPMOs, the best structural match being Serratia marscecens AA10, previously known as CBP21 (6). While the third, non-coordinating active site residue is a tyrosine (Tyr184) as in most AA9s, the positioning of Ala89 is reminiscent of AA10s . These features are reflected in the seemingly rhombic EPR spectrum of the Cu(ll) form of Td(AA14)A, which is best modelled as a mixture of two separate copper coordination geometries . The parallel values for both species could be determined accurately (species 1 , gz = 2.283, | Az | = 407 MHz; species 2, gz = 2.258, | Az | = 512 MHz) , and their ratio was shown to be dependent on pH, buffer and glycerol content. Both species fall into a Peisach-Blumberg Type 2 classification, typical of LPMOs, although the somewhat reduced | Az | value for species 1 , along with rhombic gx and gy values, shows some distortion away from axial symmetry, possibly influenced by the presence of the Ala89 side chain near the copper site. The mixed species observed and the variation in their ratio in response to exogenous ligands and pH reveal not only the accessible nature of the copper site to substrates, but also the high degree of flexibility in the copper coordination sphere, a factor which may be important in the mechanism of action.

Interestingly, on opposite sides of the histidine brace and almost perfectly mirroring each other, are the co-planar aromatic rings of Tyr166 and Tyr24, which mark the boundaries of the flat surface surrounding the active site and could be involved in substrate binding, as previously suggested for this type of residue by other authors (20). The structure of Td(AA14)A also contains chains of aromatic residues that form a path through the enzyme core and could conceivably mediate electron transfer. What is more, the surface to which these residues lead forms a negatively charged patch, which may well represent a candidate docking site for a protein partner. Our gut proteomics data indicate the presence of several putative dehydrogenases that could play the role of electron donor in T. domestica.

While having most of the canonical features found in other LMPOs, the AA14 structure reveals something entirely new. A b-tongue-like protrusion which links strands 8 and 11 forms part of the surface surrounding the active site and has not been observed in other LPMOs. To investigate whether the protrusion is a modification specific to this new family of LPMOs, we aligned the sequences of 214 family members identified in CAZy, including 21 from T. domestica, and analyzed the sequence conservation using the ConSurf server (21). This analysis highlighted the absolute conservation at the protein active site within the family but suggests that the protrusion, while found in all T. domestica LPMOs, is not necessarily conserved across the whole AA14 family. Determination of its importance for mediating substrate specificity will, therefore, require further structural and biochemical characterization of other family members.

The reported discovery of the AA14 family and its phylogenetic, biochemical and structural characterization could have far-reaching implications in terms of our understanding of evolutionary history. The identification of these new LPMOs across diverse eukaryotic Taxa, including unicellular algae, is unprecedented among characterized LPMO families and strongly suggests an ancient origin, possibly dating back to the first build-up of atmospheric oxygen roughly two billion years ago (22). Although we currently lack data to support this hypothesis, our work on T. domestica provides evidence of the importance of these LPMOs in the more recent evolution of plant cell wall digestion mechanisms in insects.

Until a few decades ago, the hypothesis that insects evolved the ability to digest plant cell wall components via symbiotic relationships with microorganisms was widely accepted. However, while the origin of obligate symbioses for plant cell wall digestion is thought to have occurred about 100 million years ago (23), the most recent phylogenomic analysis demonstrates that the earliest terrestrial insects appeared during the Ordovician, roughly 480 million years ago (Mya) (24) and thrived during the Devonian and Carboniferous. This implies that primordial detritivorous insects must have relied on endogenous enzymes for digestion of plant matter. In support of this, endogenous cell-wall degrading enzymes (cellulases, b-glucosidases, b-I ^^Iuoqhqebe, pectinases) have been reported in all major insect lineages, suggesting that the ancestral mechanisms for plant cell wall digestion in invertebrates were independent from microbial symbioses (25, 26), and in at least one case an invertebrate has been shown to live on a diet of wood without microbial assistance (27, 28).

Fossil records and phylogenomic analysis show that the order Zygentoma, which includes T. domestica, was one of the very first insect groups to appear on land, more than 400 Mya. Members of this group appear not to have evolved significantly since this ancient origin (24) and have been described as“living fossils”. By revealing the abundance of endogenous lignocellulolytic enzymes in the gut of T. domestica, our proteomic studies confirm that plant cell wall digestion by endogenous enzymes may indeed be an ancestral trait in insects. The possession of these enzymes might help explain why insects thrived during the Carboniferous period (360-286 Mya), when plants fully colonized the land and atmospheric oxygen levels reached a peak of 35% compared to the current 21%. Such conditions would have likely favored the recruitment and expansion of carbohydrate-active oxidative enzymes for the degradation of abundant biomass.

Our sequence alignments, phylogenetic analysis, structural and biochemical characterization strongly suggest that the newly identified LPMOs are key part of this ancestral mechanism and help explain why T. domestica is one of the most efficient cellulose degraders in the animal world. Furthermore, the notable boosting effect of the T. domestica LPMO on the activity of glycoside hydrolases on both cellulose and chitin, the two most abundant polysaccharides on the planet, may offer a powerful new tool for a range of biotechnological applications.

References

1. R. A. Prins, D. A. Kreulen, Comparative aspects of plant cell wall digestion in insects.

Animal Feed Science and Technology 32, 101-118 (1991)

2. D. Zinkler, M. Gotze, Cellulose digestion by the firebrat T. domestica domestica.

Comp. Biochem. Physiol. 88B, 661-666 (1987)

3. R. Lasker, A. Giese, Cellulose digestion by the silverfish Ctenoiepisma lineata. Journal of Experimental Biology 33, 542-553 (1956)

4. E. Lindsay, The biology of the silverfish, Ctenoiepisma lingicaudata Esch. with particular reference to its feeding habits. Proceedings of the Royal Society of Victoria 52, 35-83, (1940)

5. D. S. Treves, M. M. Martin, Cellulose digestion in primitive hexapods: effect of ingested antibiotics on gut microbial populations and gut cellulase levels in the firebrat, T. domestica domestica (Zygentoma, Lepismatidae). Journal of Chemical Ecology 20, 2003-2020 (1994)

6. G. Vaaje-Kolstad, B. Westereng, S. J. Horn, Z. Liu, H. Zhai, M. Sorlie, V. G. H. Eijsink,

An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides. Science 330, 219-222 (2010)

7. R. J. Quinlan, M. D. Sweeney, L. Lo Leggio, H. Otten, J. N. Poulsen, K. S. Johansen,

K. B. R. M. Krogh, C. I. Jorgensen, M. Tovborg, A. Anthonsen, T. Tryfona, C. P. Walter, P. Dupree, F. Xu, G. J. Davies, P.H. Walton, Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components. Proc. Natl. Acad. Sci. U.S.A. 108, 15079-15084 (2011)

8. G. R. Hemsworth, B. Henrissat, G. J. Davies, P. H. Walton, Discovery and characterization of a new family of lytic polysaccharide monooxygenases. Nat. Chem. Biol. 10, 122-126 (2014)

9. L. L. Leggio, T. J. Simmons, J. N. Poulsen, K. E. H. Frandsen, G. R. Hemsworth, M.

A. Stringer, P. von Freiesleben, M. Tovborg, K. S. Johansen, L. De Maria, P. V. Harris,

C. Soong, P. Dupree, T. Tryfona, N. Lenfant, B. Henrissat, G. J. Davies, P.H. Walton, Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase. Nat. Comm. 6, 5961 (2015)

10. A. J. Langston, T. Shaghasi, E. Abbate, F. Xu, E. Vlasenko, M. D. Sweeney, Oxidoreductive cellulose depolymerization by the enzymes cellobiose dehydrogenase and glycoside hydrolase 61. Appl. Environ. Microbiol. 77, 7007-7015 (2011)

11. K. S. Johansen, Discovery and industrial applications of lytic polysaccharide monooxygenases. Biochem. Soc. Trans. 44, 143-149 (2016) 12. V. Lombard, H. Golaconda Ramulu, E. Drula, P. M. Coutinho, B. Henrissat, The Carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 42, D490-D4951 (2014)

13. G. R. Hemsworth, E. M. Johnston, G. J. Davies, P. H. Walton, Lytic polysaccharide monooxygenases in biomass conversion. Trends in Biotechnol. 33, 747-761 (2015)

14. Y. Ishihama, Y. Oda, T. Tabata, T. Sato, T. Nagasu, J. Rappsilber, M. Mann, Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol. Cell Proteomics 4, 1265-1272 (2005).

15. A. B. Boraston, D. N. Bolam, H. J. Gilbert, G. J. Davies. Carbohydrate-binding modules: fine-tuning polysaccharide recognition. Biochem. J. 382, 769-681 (2004)

16. A. Kohler, A. Kuo, L. G. Nagy, E. Morin, K. W. Barry, F. Buscot, B. Canback, C. Choi, N. Cichocki, A. Clum, J. Colpaert, A. Copeland, M. D. Costa, J. Dore, D. Floudas.G. Gay, M. Girlanda, B. Henrissat, S. Herrmann, J. Hess, N. Hogberg, T. Johansson, H. Khouja, K. LaButti, U. Lahrmann, A. Levasseur, E. A. Lindquist, A. Lipzen.R. Marmeisse, E. Martino, C. Murat, C. Y. Ngan, U. Nehls, J. M. Plett, A. Pringle, R. A. Ohm, S. Perotto, M. Peter, R. Riley, F. Rineau, J. Ruytinx, A. Salamov, F. Shah, H. Sun, M. Tarkka, A. Tritt, C. Veneault-Fourrey, A. Zuccaro, Mycorrhizal Genomics Initiative Consortium, A. Tunlid, I. V. Grigoriev, D. S. Hibbett, F. Martin, Convergent losses of decay mechanisms and rapid turnover of symbiosis genes in mycorrhizal mutualists. Nat. genetics 47, 410-415 (2015)

17. G. R. Hemsworth, E. J. Taylor, R. Q. Kim, R. C. Gregory, S. J. Lewis, J. P. Turkenburg, A. Parkin, G. J. Davies, P. H. Walton, The Copper Active Site of CBM33 Polysaccharide Oxygenases. J. Am. Chem. Soc. 135, 6069-6077 (2013)

18. D. Kracher, S. Scheiblbrandner, A. K. G. Felice, E. Breslmayr, M. Preims, K.

Ludwicka, D. Haltrich, V. G. H. Eijsink, R. Ludwig, Extracellular electron transfer systems fuel cellulose oxidative degradation. Science 352, 1098-1101 (2016)

19. L. Holm, P. Rosenstrom, Dali server: conservation mapping in 3D. Nucleic Acids Res.

38, W545-549 (2010)

20. K. E. Frandsen, T. J. Simmons, P. Dupree, J. C. Poulsen, G. R. Hemsworth, L. Ciano, E. M. Johnston, M. Tovborg, K. S. Johansen, P. von Freiesleben, L. Marmuse, S. Fort, S. Cottaz, H. Driguez, B. Henrissat, N. Lenfant, F. Tuna, A. Baldansuren, G. J. Davies, L. Lo Leggio, P. H. Walton, The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases. Nat. Chem. Biol. 12, 298-303 (2016)

21. H. Ashkenazy, E. Erez, E. Martz, T. Pupko, N. Ben-Tal, ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res. 38, W529-533 (2010)

22. N. J. Planavsky, D. Asael, A. Hofmann, C. T. Reinhard, S. V. Lalonde, A. Knudsen, X.

Wang, F. O. Ossa, E. Pecoits, A. J. B. Smith, N. J. Beukes, A. Bekker, T. M. Johnson, K. O. Konhauser, T. W. Lyons, O. J. Rouxel, Evidence for oxygenic photosynthesis half a billion years before the Great Oxidation Event. Nature Geoscience 7, 283-286 (2014) 23. G. O. Poinar Jr, Description of an early Cretaceous termite (Isoptera: Kalotermitidae) and its associated intestinal protozoa, with comments on their co-evolution. Parasites & Vectors 2, 12 (2009)

24. B. Misof, S. Liu, K. Meusemann, R. S. Peters, A. Donath, C. Mayer, P. B. Frandsen, J. Ware, T. Flouri, R. G. Beutel, O. Niehuis, M. Petersen, F. Izquierdo-Carrasco, T. Wappler, J. Rust, A. J. Aberer, U. Aspock, H. Aspock, D. Bartel, A. Blanke, S. Berger,

A. Bohm, T. R. Buckley, B. Calcott, J. Chen, F. Friedrich, M. Fukui, M. Fujita, C. Greve,

P. Grobe, S. Gu, Y. Huang, L. S. Jermiin, A. Y. Kawahara, L. Krogmann, M. Kubiak,

R. Lanfear, H. Letsch, Y. Li, Z. Li, J. Li, H. Lu, R. Machida, Y. Mashimo, P. Kapli, D. D. McKenna, G. Meng, Y. Nakagaki, J. L. Navarrete-Heredia, M. Ott, Y. Ou, G. Pass, L. Podsiadlowski, H. Pohl, B. M. von Reumont, K. Schutte , K. Sekiya , S. Shimizu, A. Slipinski, A. Stamatakis, W. Song, X. Su, N. U. Szucsich, M. Tan, X. Tan, M. Tang, J. Tang, G. Timelthaler, S. Tomizuka, M. Trautwein, X. Tong, T. Uchifune, M. G. Walzl,

B. M. Wiegmann, J. Wilbrandt, B. Wipfler, T. K. Wong, Q. Wu, G. Wu, Y. Xie, S. Yang,

Q. Yang, D. K. Yeates, K. Yoshizawa, Q. Zhang, R. Zhang, W. Zhang, Y. Zhang, J. Zhao, C. Zhou, L. Zhou, T. Ziesmann, S. Zou, Y. Li, X. Xu, Y. Zhang, H. Yang, J. Wang, J. Wang, K. M. Kjer, X. Zhou, Phylogenomics resolves the timing and pattern of insect evolution. Science 346,763-767 (2014)

25. N. Calderon-Cortes, M. Quesada, H. Watanabe, H. Cano-Camacho, K. Oyama, Endogenous Plant Cell Wall Digestion: A Key Mechanism in Insect Evolution. Annu. Rev. Ecol. Evol. Syst. 43, 45-71 (2012)

26. D. D. McKenna, E. D. Scully, Y. Pauchet, K. Hoover, R. Kirsch, S. M. Geib, R. F.

Mitchell, R. M. Waterhouse, S. Ahn, D. Arsala, J. B. Benoit, H. Blackmon, T. Bledsoe,

J. H. Bowsher, A. Busch, B. Calla, H. Chao, A. K. Childers, C. Childers, D. J. Clarke,

L. Cohen, J. P. Demuth, H. Dinh, H. Doddapaneni, A. Dolan, J. J. Duan, S. Dugan, M. Friedrich, K. M. Glastad, M. A. D. Goodisman, S. Haddad, Y. Han, D. S. T. Hughes, P. loannidis, J. S. Johnston, J. W. Jones, L. A. Kuhn, D. R. Lance, C. Lee, S. L. Lee, H. Lin, J. A. Lynch, A. P. Moczek, S. C. Murali, D. M. Muzny, D. R. Nelson, S. R. Palli,

K. A. Panfilio, D. Pers, M. F. Poelchau, H. Quan, J. Qu, A. M. Ray, J. P. Rinehart, H.

M. Robertson, R. Roehrdanz, A. J. Rosendale, S. Shin, C. Silva, A. S. Torson, I. M. Vargas Jentzsch, J. H. Werren, K. C. Worley, G. Yocum, E. M. Zdobnov, R. A. Gibbs,

S. Richards, Genome of the Asian longhorn beetle ( Anopiophora glabripennis), a globally significant invasive species, reveals key functional and evolutionary innovations at the beetle-plant interface. Genome Biology 17, 227 (2016)

27. A. J. King, S. M. Cragg, Y. Li, J. Dymond, M. J. Guille, D. J. Bowles, N. C. Bruce, I. A.

Graham, S. J. McQueen-Mason, Molecular insight into lignocellulose digestion by a marine isopod in the absence of gut microbes. Proc. Natl. Acad. Sci. U.S.A. 107, 5345- 5350 (2010)

28. M. Kern, J. E. McGeehan, S.D. Streeter, R. N. A. Martin, K. Besser, L. Elias, W.

Eborall, G. P. Malyon, C. M. Payne, M. E. Himmel, K. Schnorr, G. T. Beckham, S. M. Cragg, N. C. Bruce, S. J. McQueen-Mason, Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance. Proc. Natl. Acad. Sci. U.S.A. 110, 10189-10194, (2013)

29. A. A. Dowle, J. Wilson, J. R. Thomas, Comparing the Diagnostic Classification Accuracy of iTRAQ, Peak-Area, Spectral-Counting, and emPAI Methods for Relative Quantification in Expression Proteomics. J. Proteome Res. 10, 3550-3562 (2016)

30. Y. Ishihama, Y. Oda, T. Tabata, T. Sato, T. Nagasu, J. Rappsilber, M. Mann, Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol Cell Proteomics. 4, 1265-1272 (2005).

31. R. C Edgar, MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792-1797 (2004)

32. S. Henikoff, J. G. Henikoff, Amino-acid substitution matrices from protein blocks. Proc.

Natl. Acad. Sci. USA 89, 10915-10919 (1992)

33. N. Saitou, M. Nei, The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 4, 406-425 (1987)

34. D. H. Huson, D. C. Richter, C. Rausch, T. Dezulian, M. Franz, R. Rupp, Dendroscope:

An interactive viewer for large phylogenetic trees. BMC Bioinformatics 8, 460 (2007)

35. R. T. Byrne, F. Whelan, P. Aller, L. E. Bird, A. Dowle, C. M. Lobley, Y. Reddivari, J. E.

Nettleship, R. J. Owens, A. A. Antson, D. G. Waterman S-Adenosyl-S-carboxymethyl- L-homocysteine: a novel cofactor found in the putative tRNA-modifying enzyme CmoA. Acta Crystallogr D Biol Crystallogr. 69,1090-1098 (2013)

36. S. Abdul Rahman, E. Bergstrom, C. J. Watson, K. M. Wilson, D. A. Ashford, J. R.

Thomas, D. Ungar, J. E. Thomas-Oates. Filter-aided N-glycan separation (FANGS): a convenient sample preparation method for mass spectrometric N-glycan profiling. J. Proteome Res. 13, 1167-1176 (2014)

37. 1. Ciucanu, F. Kerek, A simple and rapid method for permethylation of carbohydrates.

Carbohydrate Res. 131 , 209-217 (1984)

38. B. Domon, C. E. Costello, A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates. Glycoconj. J. 5, 397-409 (1988)

39. W. Kabsch, XSD. Acta Crystallogr D Biol Crystallogr. 66, 125-132 (2010)

40. M. D. Winn, C. C. Ballard, K. D. Cowtan, E. J. Dodson, P. Emsley, P. R. Evans, R. M.

Keegan, E. B. Krissinel, A. G. Leslie, A. McCoy, S. J. McNicholas, G. N. Murshudov, N. S. Pannu, E. A. Potterton, H. R. Powell, R. J. Read, A. Vagin, K. S. Wilson, Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr. 67, 235-242 (2011)

41. J. Foadi, P. Aller, Y. Alguel, A. Cameron, D. Axford, R. L. Owen, W. Armour, D. G.

Waterman, S. Iwata, G. Evans, Clustering procedures for the optimal selection of data sets from multiple crystals in macromolecular crystallography. Acta Crystallogr D Biol Crystallogr. 69, 1617-1632 (2013)

42. G. M. Sheldrick, A short history of SHELX. Acta Crystallogr A. 64, 112-122 (2008)

43. K. Cowtan, The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr D Biol Crystallogr. 62, 1002-1011 (2006)

44. P. Emsley, K. Cowtan, Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr. 60, 2126-2132 (2004)

45. G. N. Murshudov, A. A. Vagin, E. J. Dodson, Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr. 53, 240-255 (1997) 46. 1. W. Davis, A. Leaver-Fay, V. B. Chen, J. N. Block, G. J. Kapral, X. Wang, L. W. Murray, W. B. Arendall 3rd, J. Snoeyink, J. S. Richardson, D. C. Richardson, MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375-383 (2007)

47. K. Katoh, D. M. Standley, MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evoi. 30, 772-780 (2013) 48. H. Ashkenazy, E. Erez, E. Martz, T. Pupko, N. Ben-Tal, ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res. 38, W529-533 (2010)

49. S. Stoll, A. Schweiger, EasySpin, a comprehensive software package for

spectral simulation and analysis in EPR. J. Magn. Resort. 178, 42-55 (2006)

50. Inc., T.M. MATLAB and Statistics Toolbox Release 2014a. (The MathWorks,

Inc., Natick, Massachusetts, United States)

1.

Claims

Claims

1. An inhibitory agent comprising an inhibitory RNA wherein said inhibitory RNA comprises an antisense nucleotide sequence complementary to a sense RNA molecule that encodes a lytic polysaccharide monooxygenase.

2. The agent according to claim 1 comprising an inhibitory RNA complementary to a sense RNA molecule encoding a fungal lytic polysaccharide monooxygenase.

3. The agent according to claim 1 comprising an inhibitory RNA complementary to a sense RNA molecule encoding an oomycete lytic polysaccharide monooxygenase.

4. The agent according to any one of claims 1 to 3 wherein said agent comprises an inhibitory RNA that is complementary to a part of a sense nucleic acid molecule selected from the group consisting of: i) a nucleic acid molecule comprising a nucleotide sequence set forth in SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43 or 44; ii) a nucleic acid molecule that is at least 75% identical to the nucleotide sequence set forth in SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43 or 44 and that encodes a polypeptide with lytic polysaccharide monooxygenase activity.

5. The agent according to any one of claims 1 to 3 wherein In a preferred embodiment of the invention said agent comprises an inhibitory RNA that is complementary to a part of a sense nucleic acid molecule selected from the group consisting of: i) a nucleic acid molecule comprising a nucleotide sequence set forth in SEQ ID NO: 55, 56, 58, 59, 61 , 62, 64, 65, 67, 68, 70, 71 , 73, 74, 76, 77, 79, 80, 82, 83, 85, 86, 88, 89, 91 or 92; ii) a nucleic acid molecule that is at least 75% identical to the nucleotide sequence set forth in SEQ ID NO: 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67 and that encodes a polypeptide with lytic polysaccharide monooxygenase activity.

6. The agent according to any one of claims 1 to 3 wherein said agent comprises an inhibitory RNA that is complementary to a part of a sense nucleic acid molecule selected from the group consisting of: i) a nucleic acid molecule comprising a nucleotide sequence set forth in SEQ ID NO: 68, 69, 70, 71 , 72, 73, 74. 75. 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 ii) a nucleic acid molecule that is at least 50% identical to the nucleotide sequence set forth in SEQ ID NO: 68, 69, 70, 71 , 72, 73, 74. 75. 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 and that encodes a polypeptide with lytic polysaccharide monooxygenase activity.

7. The agent according to any one of claims 1 to 3 wherein said agent comprises an inhibitory RNA that is complementary to a part of a sense nucleic acid molecule selected from the group consisting of:

i) a nucleic acid molecule comprising a nucleotide sequence set forth in SEQ ID NO:103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113 or 114; ii) a nucleic acid molecule that is at least 50% identical to the nucleotide sequence set forth in SEQ ID NO:103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113 or 114; and that encodes a polypeptide with lytic polysaccharide monooxygenase activity.

8. The agent according to any one of claims 1 to 3 wherein said agent comprises an inhibitory RNA that is complementary to a part of a sense nucleic acid molecule selected from the group consisting of:

i) a nucleic acid molecule comprising a nucleotide sequence set forth in SEQ ID NO: 115, 116, 117, 118, 119 or 120;

ii) a nucleic acid molecule that is at least 50% identical to the nucleotide sequence set forth in SEQ ID NO: 115, 116, 117, 118, 119 or 120and that encodes a polypeptide with lytic polysaccharide monooxygenase activity.

9. The agent according to any one of claims 1 to 8 wherein In a preferred embodiment of the invention said inhibitory RNA that is complementary to said part of said sense nucleic acid molecule comprises or consists of between 19 and 29 nucleotides.

10. A composition comprising an agent according to any one of claims 1 to 9 adapted for spray application of the agent,

11. A nucleic acid molecule comprising a transcription cassette wherein said cassette includes a nucleotide sequence of an inhibitory RNA that inhibits the expression of a lytic polysaccharide monooxygenase wherein said cassette is adapted for expression by provision of at least one promoter operably linked to said nucleotide sequence such that both sense and antisense molecules are transcribed from said cassette thereby expressing said inhibitory RNA.

12. A vector comprising a transcription cassette according to claim 11 wherein the cassette is part of an expression vector suitable for transformation of a eukaryotic cell.

13. The vector according to claim 12 wherein said eukaryotic cell is a plant cell.

14. The vector according to claim 12 wherein said eukaryotic cell is a fungal or oomycete cell.

15. A plant transformed with the transcription cassette or expression vector according to claim 12 or 13.

16. A fungal or oomycete cell transformed with a transcription cassette or expression vector according to claim 12 or 13.

17. The use an inhibitory agent comprising an inhibitory RNA wherein said inhibitory RNA comprises an antisense nucleotide sequence complementary to an RNA molecule that encodes a lytic polysaccharide monooxygenase in the control of eukaryotic microbial pathogens.

18. The use according to claim 17 wherein said eukaryotic microbial pathogen is a fungal or oomycete pathogen.

19. The use according to claim 18 wherein said fungal pathogen is a plant fungal pathogen.

20 The use according to claim 19 wherein said fungal pathogen is Botrytis.

21. The use according to claim 20 wherein said fungal pathogen is Botrytis cinerea.

22. The use according to claim 18 wherein said eukaryotic microbial pathogen is an oomycete pathogen.

23. The use according to claim 22 wherein said eukaryotic microbial pathogen is a plant oomycete pathogen.

24. The use according to claim 23 wherein said plant oomycete pathogen s Phtythophora.

25. A method for the control of eukaryotic pathogenic microbial species comprising the steps: i) formulating a composition comprsing an agent according to any one of claims 1 to 9 or a composition according to claim 10; and ii) application of the composition to a plant or part of a plant to be treated.