Molecular methods for studying the Cryphonectria parasitica – hypovirus experimental system.

Molecular methods for studying the Cryphonectria parasitica – hypovirus experimental system.

Angus L. Dawe*, 1, 2 , Rong Mu2, Gloricelys Rivera1 and Joanna A. Salamon1.

1

Department of Biology and 2Molecular Biology Program, New Mexico State University, Las

Cruces, NM 88003

*Corresponding Author: Department of Biology, New Mexico State University, MSC 3AF, PO Box 30001 Las Cruces, NM 88003. [email protected] Phone: 575-646-4003 Fax: 575-646-5665

(i) Abstract The interaction of the filamentous fungal plant pathogen Cryphonectria parasitica with its virulence-attenuating viruses provides a unique platform to explore the molecular biology and genetics of virus-host interactions. Following the development of transformation procedures for this fungus, subsequent advances include infectious cDNA clones of several members of the Hypoviridae and an imminently complete fungal genome project. Presented here are basic protocols for growth of the organism and the extraction of DNA, RNA and protein. Additionally, two further protocols are provided for investigations of host protein phosphorylation and for viral genome secondary structure.

Key Words Hypovirulence, protein phosphorylation, phosphatase, RNA secondary structure, RNase digestion

1. Introduction

This review focuses on techniques used in the study of Cryphonectria parasitica, a filamentous fungal plant pathogen, and its associated virulence-attenuating mycovirus. C. parasitica is a member of the phylum Ascomycota and the causative agent of chestnut blight. First observed in the United States in the early part of the 20th century (1), the fungus rapidly spread throughout the natural range of Castanea dentata, the American chestnut, resulting in the near-eradication of this species. The blight also appeared in Europe during the 1930s (2), affecting the European chestnut, Castanea sativa. However, the observation of healing trees (3) led to the isolation of hypovirulent strains of C. parasitica (4) that were subsequently shown to contain a double-stranded RNA (dsRNA) species (5, 6), and recognized as a new family mycoviruses, the Hypoviridae (7). The single most striking phenotype that these viruses impose upon the fungal host is a reduction in pathogenesis. Additional phenotypes are noted in laboratory cultures of hypovirulent strains, including reduced pigmentation, asexual sporulation and radial growth rate, altered colony morphology and female sterility (reviewed by (8)). The phenomenon of hypovirulence provides potential for biological control of the chestnut blight fungus (9). The hypovirus genome of the CHV1 species consists of a 12.7 kb dsRNA molecule (10) expressed in two open reading frames. The smaller ORF A encodes p69, which gives rise to two polypeptides, p29 and p40 via an autocatalytic event (11). The polyprotein of ORF B is less well characterized, with a single event known to liberate p48 from the N-terminus (10). Other mature protein products are as yet uncharacterized. Most closely related to positive-strand RNA viruses of the potyvirus group (12), the family Hypoviridae represent a unique experimental system that permits in-depth analysis

using molecular tools to genetically modify both a host (C. parasitica) and its parasite (the mycovirus) and to observe further interactions with a third organism (the chestnut). It is then possible to examine the effects of these changes on both the host phenotype and virulence on the chestnut. A protoplast-based protocol for transformation of C. parasitica was developed in 1990 (13) and is still essentially used unchanged. Presented here are basic methods to approach different aspects of the molecular biology of the mycovirus-host interaction (general procedures for isolation of protein, DNA and RNA) and two more detailed protocols we have used to examine host protein phosphorylation and viral genome secondary structure.

2. Materials 2.1 Growth and harvesting of fungal strains (see Note 1) 1. Strains of C. parasitica including the most widely used “wild type” strain (EP155) and its isogenic counterpart infected with hypovirus CHV1-EP713 are available from the ATCC culture collection, # 38755 and #52571, respectively. 2. Potato dextrose broth, 24 g / L (PDB; Difco, BD Biosciences) 3. Potato dextrose agar, 37 g / L (PDA; Difco, BD Biosciences) 4. A flat surface that can be illuminated on an approximately 12 h light / dark cycle and at an irradiance level of approximately 20 – 60 µmol s-1m-2. This can be verified with a radiometer (e.g model HD 2302, Hotek Technologies). Room temperature is usually sufficient. 5. Hand-held tissue homogenizer (e.g. Polytron PT1600E, Kinematica Inc) 6. Cellophane circles, cut by hand to match the diameter of the petri plates being used. These should be first submerged in water and then autoclaved. Store submerged at 4 °C.

7. Miracloth (EMD Biosciences) cut to fit Buchner funnel. For procedures requiring additional culturing after filtration, Miracloth can be autoclaved if wrapped lightly in aluminum foil. Autoclaving is not generally necessary for procedures where cell lysis immediately follows harvesting.

2.2. Extraction of Proteins (see Notes 2, 3 and 4) 1. Extraction Buffer: 100 mM Tris-HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl store at room temperature. 2. For protein isolation, prior to use add DTT to 10 mM, CHAPS to 1% and yeast protease inhibitor cocktail (Sigma-Aldrich) at 50 µl per gram of mycelium. 3. Liquid nitrogen. 4. Mortar and pestle.

2.3. Extraction of Total RNA. 1. For total RNA isolation, use the Plant RNA Isolation Aid, RNAqueous and DNAfree treatment kits (all from Applied Biosystems / Ambion; see Notes 5 and 6).

2.4. Extraction of Genomic DNA. 1. For genomic DNA isolation, supplement Extraction Buffer in Section 2.2 Step 1 with Triton X-100 or SDS at 2 % in place of the components in Section 2.2 Step 2 and prepare additional reagents: a. Buffer saturated Phenol / Chloroform / Isoamyl alcohol, pH 7.9 (available premixed from Applied Biosystems / Ambion).

b. Chloroform. c. 3M Sodium Acetate, pH 5.2. d. 100% Ethanol; 70 % (by volume) ethanol in water. e. TE buffer (10 mM Tris, 1 mM EDTA, pH to 7.0 with HCl). f. RNase Cocktail (Applied Biosystems / Ambion).

2.3. De-phosphorylation and Casein Kinase II-Mediated Re-phosphorylation 1. Calf Intestinal Alkaline Phosphatase (Invitrogen) 2. Adenosine triphosphate (Sigma-Aldrich) 3. Phosphatase inhibitor sodium orthovanadate (Sigma-Aldrich) 4. CK2 inhibitor DMAT, 2-dimethylamino-4, 5, 6, 7-tetrabromo-1H-benzimidazole (EMD Biosciences) 5. Whole protein extracts of strains to be tested.

2.6. In vitro transcription of viral cDNA Clones, RNase treatment and Sequencing 1. Plasmids bearing cDNA clones of the hypoviruses CHV1-EP713 (pLDST; (14)) or CHV1Euro7 (pTE7; (15)) kindly provided by Don Nuss, University of Maryland Biotechnology Institute. 2. SpeI restriction endonuclease, BSA (10 mg / ml) and NEBuffer 2 (New England Biolabs). 3. AmpliCapTM T7 and SP6 high yield message maker kit components (Epicenter Biotechnologies). 4. RNeasy Mini Kit components (QIAGEN). 5. RNase T1, RNase A, RNase I and RNase V1 (Applied Biosystems / Ambion).

6. Water baths at 50 °C and 65 °C. 7. GlycoBlue coprecipitant (Applied Biosystems / Ambion) for visualization of the RNA pellet. 8. 80 % Ethanol (by volume) in RNase-free water. 9. Eppendorf Vacufuge concentrator. 10. IR-700 labeled primer, 5’-CCACTGTAGTAGGATCAAC-3’ (see Note 7; Li-Cor Biosciences). 11. SuperScript III Reverse Transcriptase, RNaseOUT RNase inhibitor, First-strand buffer, dithiothreitol (DTT; 0.1 M), dNTP mixture (10 mM each dATP, dGTP, dTTP and dCTP), all from Invitrogen. 12. Sequitherm Excell II DNA sequencing kit (Epicenter Technologies). 13. Access to a Li-Cor 4200 DNA sequencer.

3. Methods 3.1 Growth and Harvesting of Mycelium 1. Solid-medium cultures are grown by inoculating a standard petri dish containing potato dextrose agar medium with a small (~ 2 mm x 2 mm) plug from the actively growing edge of a previously grown culture. Plates are best grown under the conditions noted above for temperature and light level. 2. For harvesting solid-grown mycelium, recovery is easiest if autoclaved cellophane is first placed on the surface of the medium after it has solidified. Once inoculated, the mycelium will grow on the cellophane but will not penetrate into the agar. The fungal tissue can then be easily recovered by scraping the mycelium off the cellophane.

3. For liquid cultures, several plugs of mycelium from a Petri plate are used to inoculate approximately 10 ml of potato dextrose broth. The cultures are left stationary at room temperature, but may be agitated daily with vigorous shaking or brief vortexing. After 3 – 4 days, the fungal mass should be homogenized using a handheld homogenizer to break up the mycelial clumps. An equal volume of fresh medium is then added and the culture incubated an additional 3 – 4 days. 4. Harvesting of the liquid cultures is best achieved by filtration. Place four layers of Miracloth into the Buchner funnel and slowly pour the culture onto the surface while applying a vacuum. The resulting mycelial pad can be washed with water and then compressed between paper towels to remove excess moisture.

3.2. Extraction of Proteins 1. Pulverize harvested mycelium under liquid nitrogen with a pestle and mortar, then carefully weigh. Transfer the ground mycelium to microfuge tube(s) and add 1.5 times the volume by weight (e.g 100 mg ground mycelium will required 150 µl) of freshly prepared protein extraction buffer with DTT, CHAPS and protease inhibitors. 2. Vortex vigorously and incubate on ice for 15 minutes. Repeat vortexing and incubate a further 15 minutes on ice. 3. Pellet the cellular debris for 5 minutes at 4 °C and maximum speed in a microcentrifuge. 4. Carefully pipet the clear protein lysate and transfer to a clean microfuge tube. Avoid aspirating the pellet. If the lysates are not clear repeat the centrifugation step. 5. Determine protein concentration using the Bradford Assay system from BioRad. This assay is not sensitive to the concentrations of CHAPS used in this extraction method.

6. The samples can be stored at –20 °C for short-term use, but appear more stable at –80 °C for longer-term.

3.3. Extraction of Total RNA. 1. For extraction of RNA, begin by harvesting and pulverizing the mycelium as described for the preparation of proteins. Resuspend the powdered tissue in the Lysis / Binding solution from the RNaqueous kit and add the Plant RNA Isolation Aid. Use a ratio of 0.2 g ground mycelium per 1.6 ml Lysis / Binding solution and 0.2 ml Plant RNA Isolation Aid. 2. Continue with the RNaqueous kit exactly as described in the manufacturer’s literature. Elute the RNA in 60 µl Elution Solution preheated to 80 °C. Remove any DNA from the recovered sample by following the DNAfree rigorous treatment protocol. Perform this procedure twice to ensure removal of all DNA. 3. After DNA digestion, validate quality of the preparation by checking on a spectrophotometer at 260 / 280 nm. Based on the values obtained, load and run approximately 1 – 2 µg on a standard 0.8 – 1 % agarose gel. Be sure to first clean the gel box thoroughly with RNaseZap solution. When visualized by staining with Ethidium Bromide or SYBR Safe (Invitrogen), the two bands corresponding to the ribosomal RNA species should be clearly visible and any degradation products (seen as a fast-migrating species at the bottom of the gel) should be minimal or absent. Use immediately or store at –80 °C.

3.4 Extraction of Genomic DNA.

1. For extraction of genomic DNA in volumes convenient for microfuge tubes, resuspend approximately 100 µg powdered mycelium in 1.5 volumes of cold Extraction Buffer with Triton X-100 and vortex vigorously. 2. Add an equal volume of alkaline Phenol / Chloroform / Isoamyl alcohol (P/C/IAA), vortex vigorously and microfuge at maximum speed for 5 minutes at 4°C. Recover the upper aqueous layer and repeat the extraction with P/C/IAA before performing a third extraction, but with an equal volume of chloroform alone. 3. Carefully aspirate the aqueous layer, add 1/10 volume of 3M Sodium Acetate, pH 5.2, and 2 volumes of 100% Ethanol. Precipitate for at least 30 minutes (but may be left overnight at –20 °C. Pellet DNA in a microfuge for 20 minutes at maximum speed and 4 °C. Aspirate the ethanol with vacuum or pipetor and leave the pellet to air-dry. Carefully rinse the pellet with 70 % ethanol to reduce the salt content, and re-pellet. 4. Resuspend the pellet in 100 µl TE buffer and add 5 µl of the RNase Cocktail. Incubate 30 minutes at room temperature then verify the yield of your DNA by spectrophotometry.

3.5. De-phosphorylation and Re-phosphorylation of proteins.

Phosphorylation by Protein Kinase 2 (CK2) is a common modification that influences a wide array of cellular signal transduction pathways. To confirm whether there are any physiologically relevant CK2 phosphorylation sites within a protein, the covalently bound phosphates can first be removed by in vitro treatment with Calf Intestinal Alkaline Phosphatase (CIAP). This enzyme is the same routinely used for modification of DNA during certain cloning procedures and, in fact, is purchased from the supplier (Invitrogen) prepared for that purpose. We have analyzed the

phosphorylation state of the phosducin-like protein BDM-1 using antibodies raised specifically against this protein for the western blot. While this protocol was designed to analyze the modification of BDM-1 by CK2, if the protein of interest is suspected to be the target of a different kinase, other pharmacological agents are available that could be applied.

1. For protein dephosphorylation, dilute protein lysate to concentration of 1 µg / µl with dilution buffer supplied by CIAP enzyme manufacturer. Aliquot 35 µl of protein lysate, 4 µl of 10X reaction buffer provided with the enzyme and 1 µl (20,000 units) of CIAP. Incubate the reaction mixture for 30 minutes at 37 ºC. 2. De-phosphorylated proteins are re-phosphorylated by addition of equal volume of protein extract in presence of 1 mM ATP, 100 mM sodium orthovanadate (to inhibit residual CIP activity) and 20 µM specific Casein Kinase II (CK2) inhibitor DMAT (16). The reaction is performed at room temperature in the dark for 22 hours. 3. The presence or absence of the charged phosphate moiety on the protein of interest should be detectable as differential migration after conventional polyacrylamide electrophoreisis and transfer to a nylon membrane for western blotting. For BDM-1, we have successfully used NuPAGE 10 % Bis-Tris Gels (Invitrogen) in MOPS running buffer (50 mM MOPS, 50 mM Tris base, 0.1 % SDS, 1 mM EDTA, pH 7.7) and a BioRad transblot semi-dry apparatus for transfer to Immobilon-P nylon membranes from Millipore. An affinity-purified polyclonal antiserum raised in rabbits against the entire protein and a horseradish peroxidase-conjugated anti-rabbit secondary antibody from BioRad completes the blotting procedure. Optimal separation parameters to view the subtle changes in migration may vary according to protein analyzed.

3.6. RNase mapping of structural features in the hypovirus genome. Structural features of viral RNA genomes have been shown to be important for aspects of translation and viral genome replication (17, 18). In an effort to better understand the components of the hypovirus genome that are required for maintenance of the hypovirus-infected phenotype in the host mycelium, we have modified available protocols for RNA structure analysis that analyze the products of specific degradation by individual RNase activities. This approach has several distinct stages: the generation of an in vitro transcript from an available cDNA clone, RNase digestion and reverse transcription of the resulting RNA fragments resulting in the incorporation of a labeled primer.

1. Linearize the cDNA clone by digesting 2.5 µg DNA with SpeI in a 10 µl reaction with 1 µl of NEBuffer 2 and 1 mg / ml BSA at 37 °C for 90 minutes. Confirm the concentration of the product by spectrophotometer after linearization. 2. Using the materials provided in the AmpliCapTM T7 and SP6 high yield Message Maker kit, transcribe 1 µg of the linearized cDNA clone in a reaction volume that includes 2 µl Amplicap-Max transcription buffer, 2 µl 100 mM DTT, 8 µl Amplicap-Max Cap/NTP Premix, 2 µl Amplicap-Max T7 enzyme and water to 20 µl. Incubate at 37 °C for 2 hours. 3. Use the RNeasy mini kit to purify the transcripts, eluting in 30 µl RNase-free water. Check the concentration of the RNA by spectrophotometer, then store at –80 °C. 4. Dilute the RNases in water to the following concentrations: 0.1 U / µl of RNase T1, 0.02 U / µl of RNase A, 4 U / µl of RNase I and 0.01 U / µl of RNase V1 (see Note 8).

5. Add 4 µg RNA sample from Step 3, 4 µl of 10X RNA structure buffer and 0.3 µl yeast RNA to a microfuge tube. Add RNase-free water to make the total volume 40 µl. Incubate at 65°C for 2 minutes, then cool to room temperature. 6. Divide the 40 µl from Step 5 into four microfuge tubes. Add 1 µl of a different diluted RNase to each tube and incubate at room temperature for 10 minutes. Stop the reaction by addition of inactivation/precipitation buffer to each tube. 7. Add 2 µl Glycoblue to each tube and incubate the tubes at – 20 °C for 15 minutes before pelleting in a microcentrifuge at maximum speed for 15 minutes. 8. Discard the supernatant. Add 200 µl 80% ethanol to each pellet, then re-centrifuge as in Step 7. Carefully aspirate the ethanol and dry the pellets for 20 minutes under vacuum in a Vacufuge concentrator. Resuspend the dried pellets in 10 µl of RNase-free water. 9. Add reverse transcription reagents to each tube and make the total volume 20 µl: 1 µl RNase inhibitor, 5 µl 5X First Strand buffer, 1 µl DTT (0.1 M), 1 µl dNTP mixture (10 mM each), 1 µl Licor primer (5 pM) and 1 µl Superscript III Reverse Transcriptase. Cover the tube with foil and incubate the tubes at 50 °C for 1 hour. The reactions are stored at –20 °C for later analysis. 10. Control sequencing reactions are prepared by adding 1 µg cDNA clone (plasmid or linearized), 1 µl of Licor primer (2.5pM), 7.2 µl of 3.5X buffer and 1µl of polymerase from the Sequitherm sequencing kit, with RNase-free water to a total volume 20 µl to a microfuge tube. Keeping the tube on ice, divide the 20 µl mixture into four thin-wall PCR tubes and add 2 µl of terminator mix A (or T or C or G) from the sequencing kit to each PCR tube.

11. Perform the PCR with the following parameters: 95°C for 3 minutes followed by 30 cycles of 95°C for 30 seconds, 53°C for 30 seconds and 70°C for 1 minute. Add 3 µl of the Stop solution provided and store the reactions at –20 °C wrapped in foil. 12. The individual RNase-treated reactions, the control sequencing reaction and untreated negative controls must now be separated using the Li-Cor sequencer according to the protocols and guidelines of the facility you are working with. 13. From the resulting gel image, the location of the bands in the RNase treatment lanes represents a cleavage event. These are located in the sequence as a whole by reading the sequence control reaction from the bottom of the gel upwards. Cleavage by RNase V1 occurs at double-stranded nucleotides, by RNase I at any single stranded nucleotides, by RNase A at single-stranded C or U and by RNase T1 at single-stranded G. A small portion of a typical gel is shown in Fig. 1. By locating the sites at which there is a known feature as defined by the RNase product, it is possible to constrain a model for the secondary structure of the RNA molecule using experimentally determined characteristics.

3.7. Analyzing Secondary Structural Motifs in the Hypovirus Genome: Mfold Prediction 1. Go to web server http://mfold.bioinfo.rpi.edu/cgi-bin/rna-form1-2.3.cgi (19) to input the RNA sequence (see Note 9). 2. For C. parasitica hypoviruses, use the prediction temperature of 25 °C, the closest approximation to the actual temperature at which the organism is grown in the laboratory. Other settings are left as defaults. Do not enter any constraint information at this time. 3. After selecting the “fold RNA” button at the bottom of the screen, the input sequence may generate many alternative secondary structure prediction results. Choose the lowest free

energy for the most stable (likely) structure. This represents the baseline structure assuming no constraints imposed by the interaction of individual bases (see Fig. 1(A)). 4. Based on the RNase mapping results, repeat 1 – 3 above but include constraints that do not match the experimental observations concerning the locations of known single-stranded regions. Repeat the folding analysis to generate a secondary structure that includes experimentally validated constraints (see Fig. 1 for an example).

4. Notes 1. Growth conditions can affect the phenotype of C. parasitica, particularly excessive light or heat. Generally, stable colony morphologies can be maintained at about 21 – 24 °C. Under higher light intensity than indicated, sporulation and pigmentation may be increased. However, this can begin to ameliorate some effects of virus infection, as noted by Hillman et al (20). 2. All solutions and media should be prepared with purified water (18.2 MΩ-cm). Additionally, for critical procedures and particularly those involving RNA, RNase-free water should be used. This is most easily obtained from many suppliers (e.g. Applied Biosystems, Sigma, Fisher etc) but can also be home-made according to protocols found in Sambrook (21). 3. Traditionally, Cryphonectria extracts of DNA, RNA and protein have been generated by the method presented here – using liquid nitrogen to freeze the sample followed by manual grinding. However, the use of lyophilized mycelium has significant promise and may provide a viable alternative, especially when working with a large number of samples. After harvesting, mycelial samples (liquid or solid medium grown) can be lyophilized overnight following which the mycelium is easily powdered by agitation at room temperature using a

pipet tip, micro-homogenizer or by vortexing with acid-washed glass beads. The extraction procedure then continues as described above. 4. The method presented here is derived from an analysis of the suitability of different extraction methods for recovering G-protein signaling components described by Parsley et al. (22). Alternative extraction buffers may be found in that paper. 5. For all RNA isolation and handling procedures, be sure to clean the work area thoroughly with an RNase-decontaminating agent (e.g RNase Zap, Applied Biosystems). Wear gloves and change regularly, especially if the gloves are removed during a pause in the process. Use microfuge tubes and pipet tips that are certified as “DNase and RNase free” by the manufacturer. 6. Cryphonectria mycelium, particularly in liquid culture, produces excessive carbohydrates that interfere with many column-based isolation protocols. The manufacturer kit indicated is targeted towards isolation of RNA from plant material and works reproducibly well if all guidelines concerning biomass are observed. Other kits may also be equally effective. 7. The primer should be designed to anneal at least 40 – 50 nucleotides from the target sequence to be analyzed. The primer described above is suitable for examining the 5’ non-translated regions of the CHV1-EP713 and CHV1-Euro7 hypovirus genomes. 8. The exact concentration of RNase used was determined by viewing results after sequencing. Too little RNase results in faint or absent bands, too much precludes the resolution of bands that are close together. Unfortunately, for other viral templates these values should be taken only as a guide and a certain amount of trial and error will likely be required.

9. Alternate versions of the mFold software are available. This link directs the user to an older version of the software, but one where the temperature field is variable. For newer versions of the software the temperature field is fixed at 37 °C.

Acknowledgements This work was supported by NSF award MCB-0718735 (to ALD). GR was supported by a fellowship from the NMSU-NIH MBRS-RISE program (R25GM061222).

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Figure Legends. Figure 1. An example of experimental constraints applied to a predicted RNA structure. (A) This represents the first 48 nucleotides of the 5’ and of the untranslated region of CHV1-Euro7, as predicted by mFold with no constraints. The boxes residues indicate where the predicted structure contradicts the experimental analysis. (B) The RNase digest shows that the template was digested by RNase T1 at G26 and G35, and by RNase A at U41 and U43. These cleavage sites are also supported by the bands seen in the RNase I digest, indicated by (+). Lanes T1, A, I and V1 refer to the RNases used. Lanes U, A, G and C refer to the control sequencing reaction. Intervening lanes (-) represent primer extension controls. (C) The resulting mFold prediction when the marked paired nucleotides from (A) are constrained to be unpaired.