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Chapter 3 Construction and characterization of a kiwifruit in vivo Y2H cDNA library
Proteins regulate the majority of biological functions; their activity relies exclusively on the association with nucleic acids, other proteins, lipids and small molecules via temporary or stable complexes (Uhrig, 2006). Biological processes comprising organ development, signal transduction, plant biotic and abiotic stress responses and others are the consequences of protein-protein interactions (Rossignol et al., 2006; Uhrig, 2006). In addition to those, there are processes such as protein phosphorylation, transcription, and post-translational modifications that occur as a result of the protein-protein interactions (Cui et al., 2008; Ding et al., 2009; Zhong et al., 2008). Thus, protein-protein interactions are vital for all biological processes and exploring interaction will uncover many important roles.
Of all the methods used to find protein-protein interaction, Y2H is a commonly used tool (Auerbach and Stagljar, 2005; Cao and Yan, 2013). The reason for its widespread application is that it is used in high-throughput analysis, needs no modification of experimental conditions such as protein binding and washing procedures and the interactions can be identified in vivo. However, the Y2H procedure is prone to artefacts, so it requires rigorous positive and negative controls to validate the interaction (Auerbach and Stagljar, 2005). As described in chapter two, the host target of PsaNZV-13 AvrPto5 was not identified in the candidate gene approach based Y2H assay. This outcome suggests the adoption of an unbiased approach to screen a kiwifruit cDNA library to isolate putative host targets of PsaNZV-13 AvrPto5. To achieve that goal, firstly cDNA library generation from A. chinensis ‘Hort16A’ and characterization in yeast cells is essential.
Y2H is a genetic approach used to discover a putative interaction between two proteins in yeast. In the assay, a successful interaction will result in the reformation of two domains of a transcription factor which induces the transcription of a reporter gene (Fields and Song, 1989). This method can be used to study the interaction of two known proteins or explore the interacting partners of a particular protein of interest by cDNA library screening. In the latter case, generating a quality cDNA library is pivotal for Y2H screening.
Two approaches for the development of cDNA libraries are available today; they are digestion-ligation and recombination procedures (Cao and Yan, 2013). In the digestion-ligation method, efficient restriction digestion of the vector is vital; sub-optimal digestion can cause reduced ligation efficiency and notable insert size variation in a library (Cao and Yan, 2013). Unlike the digestion-ligation procedure, the recombination method does not need restriction and ligation; instead RNA is reverse transcribed with adapter sequences at both ends. These adapter sequences correspond to the vector, and as a result, a recombination reaction between vector and template is feasible in the presence of a recombinase enzyme. Since the recombination method does not rely on restriction enzyme digestion, the presence or absence of restriction enzyme sites do not affect cDNA clone length. Further, it offers advantages of simplicity, improved transformation efficiency and ease of handling. Similarly, the occurrence of plasmid self-ligation and chimeric clones are less frequent. Hence, this method is now predominantly employed for cDNA library construction. Y2H cDNA library development through the recombination method has two options.
One is an in vitro recombination technique (an example is the Gateway® system; www.invitrogen.com) that works by assembling cDNA into a designated entry vector followed by expression in a Y2H prey vector in the presence of recombinase enzyme. Though the technique is efficient in yielding a good quality cDNA library, the downside of the technique is that it is a lengthy process and costly if the method is used solely for Y2H library construction (Burkle et al., 2005; Cao et al., 2011). An in vivo cDNA library approach is another method (an example is the Clontech® system; www.clontech.com) wherein the recombination of cDNA template, and the prey vector occurs inside prey yeast cells upon transformation. It is not only a straightforward method but requires a shorter time for construction (Cao and Yan, 2013). In addition, once the library is developed in prey yeast cells, this can be aliquotted as 1 ml cultures in several vials for long-term storage. For screening that one ml cDNA library can be used with bait yeast cells harbouring a bait of interest. These advantages were primary drivers in the selection of the in vivo library construction in this study. In this Y2H system, GAL4 DNA binding domain is generated as bait vector and GAL4 activation domain is made as prey vector. Our proteins of interest will be developed as bait and prey in the bait and prey vectors respectively and expressed in yeast cells. A potential bait and prey proteins interaction could bring DNA binding domain (bait) and activation domain (Prey) together and form a transcription factor. The bait construct is developed to have a nuclear localization signal, as a consequence, the bait and prey interaction complex will be transported to the nucleus where the transcription of the reporter genes will take place. Thereby yeast cell grows on a particular reporter media will be identified as a positive interaction.
This chapter describes in vivo construction of an A. chinensis ‘Hort16A’ cDNA library from young leaves using the Make Your Own “Mate & Plate™” Library System (Clontech®; U.S.A). Also, it describes the characterization of kiwifruit RNA, mRNA and ds cDNA qualities, and the quality assessment of the library.
Materials and Methods
Extraction of kiwifruit total RNA
Total RNA extraction from kiwifruit leaves, DNase treatment and the integrity of the extracted total RNA were performed as per the procedures described in sections 2.2.3, 2.2.4 and 2.2.5 respectively.
Isolation of poly (A) RNA from kiwifruit total RNA
Total RNA samples with RIN values over 7 were utilized for poly (A) RNA isolation. A volume of 274 µl total RNA was taken in a 1.5 ml microcentrifuge tube (Axygen Scientific; Union City, CA, U.S.A) to extract poly (A) RNA using a NucleoTrap® mRNA kit (MACHEREY-NAGEL, Germany). An equal volume of RM0 lysis buffer was added and mixed. Subsequently, 15 µl oligo (dT) latex beads were introduced and mixed thoroughly. This was incubated at 68°C for 5 minutes, then for 10 minutes at room temperature and in-between the tube was inverted every 2 minutes. The mixture was centrifuged at 2000g for 15 seconds followed by another centrifugation at 11,000g for 2 minutes in a 5424 R microcentrifuge (Eppendorf; Hamburg, Germany). The resulting supernatant was removed, oligo (dT) latex beads were resuspended in 600 µl wash buffer RM2 by gentle up and down pipetting. The resuspended oligo (dT) latex beads were transferred to a NucleoTrap® Microfilter positioned in a 1.5 ml microcentrifuge tube and centrifuged at two different speeds as described above. The oligo (dT) latex beads were collected in the NucleoTrap® Microfilter, and the flow through was discarded. The microfilter was placed in a new 1.5 ml microcentrifuge tube, and further washing was carried out as follows: 500 µl wash buffer RM3 was added into the NucleoTrap® Microfilter containing oligo (dT) latex beads, resuspended and centrifuged at two different speeds as above. The flow-through was discarded and the NucleoTrap® Microfilter was placed again in a new 1.5 ml microcentrifuge tube, and the washing step was repeated. Additional centrifugation was carried out at 11,000g for 1 minute to remove residual wash buffer. For elution, 75 µl pre-heated (68°C) RNase-free water was added to the NucleoTrap® Microfilter within a new RNase-free 1.5 ml microcentrifuge tube, and incubated at 68°C for 7 minutes. Poly (A) RNA was eluted by centrifugation at 11,000g for 1 minute.
Kiwifruit poly (A) RNA enrichment by ethanol precipitation
An ethanol precipitation method was employed to concentrate poly (A) RNA. Isolated poly (A) RNA (60 µl) was added to a sterile RNase-free 1.5 ml microcentrifuge tube. To that 1/10th volume (6 µl) 3 M sodium acetate pH 5.2, glycogen (20 µg/µl; Invitrogen, U.S.A) 1/50th volume (1.2 µl) and two volumes (120 µl) 100% ice-cold ethanol were added, mixed and incubated overnight at -20°C. The content was centrifuged at 4°C for 25 minutes at 18,407g, and the supernatant was decanted. The pellet was washed gently once with 1 ml 70% ethanol, spun down at 4°C for 10-15 minutes, and the supernatant was removed. Poly (A) RNA pellet was dried in a laminar air flow cabinet to remove residual ethanol. Finally, 3.6 µl RNase-free ultra-pure water (Invitrogen, U.S.A) was added to resuspend the pellet. The concentration and purity of the enriched poly (A) RNA were determined by Nanodrop® (NanoDrop Technologies; Rockland, DE, USA), the integrity was verified on a 0.8% agarose gel according to section 18.104.22.168.
ds cDNA synthesis for library construction
Isolated poly (A) RNA from the above section was used in ds cDNA synthesis using a Make Your Own “Mate & PlateTM library kit (User manual; Clontech, U.S.A). The following reagents were added to an RNase-free and sterile 0.2 ml PCR tube (Axygen Scientific; Union City, CA, U.S.A), 2 µl (550 ng) kiwifruit or 1 µl mouse liver (control) (1 µg) poly (A) RNA, 1 µl CDSIII oligo-dT primer (10 µM) (5’-ATTCTAGAGGCCGAGGCGGCCGACATG-d (T) 30VN-3’), 1 µl deionized water and briefly mixed. The tube was incubated at 72°C for 2 minutes in a Mastercycler® PCR machine (Eppendorf; Hamburg, Germany), cooled on the ice briefly and spun down at 18,407g for 10 seconds in a microcentrifuge. To this, 2 µl first strand buffer (5x), 1 µl DTT (100 mM), 1 µl dNTP mix (10 mM) and 1 µl SMART MMLV (Moloney Murine Leukemia Virus) reverse transcriptase (200 unit/µl) were included, incubated for 10 minutes at 42°C then placed on ice. Subsequently, one more oligo primer known as SMART III-modified oligo (5’-AAGCAGTGGTATCAACGCA GAGTGGCCATTATGGCCGGG-3’) 1 µl (10 µM) was included in the reaction, and the incubation continued for 1 hour at 42°C. First strand synthesis reaction was stopped by heating the reaction tube to 75°C for 10 minutes. When the PCR tube was returned to room temperature (20°C), 1 µl (2 unit/µl) RNase H was added and incubated for 20 minutes at 37°C to remove residual poly (A) RNA. Long distance PCR (LD-PCR) was used to amplify ds cDNA. In total three reactions were performed, two for kiwifruit and one for control. An LD-PCR reaction of 100 µl was prepared for each sample in a sterile 0.2 ml PCR tube adding 2 µl kiwifruit or mouse first strand cDNA, 70 µl deionized water, 10 µl advantage 2 PCR buffer (10x), 2 µl dNTP mix (50x), 2 µl (10 µM/ µl) 5’ PCR primer (5’-TTCCACCCAAGCAGTGGTATCAACGCAGAGTGG-3’), 2 µl (10 µM/ µl) 3’ PCR primer (5’-GTATCGATGCCCACCCTCTAGAGGCCGAGGCGGCCGACA-3’), 10 µl melting solution (10x) and 2 µl advantage 2 polymerase mix (50x). LD-PCR was completed in the Mastercycler® PCR machine by adopting the following regime: initial denaturation at 95°C for 30 seconds, afterwards 18 cycles of amplification comprising denaturation at 95°C for 10 seconds, extension at 68°C for 6 minutes, but, each extension step was increased 5 seconds in the subsequent cycles and a final elongation for 5 minutes at 68°C. From the resulting PCR reactions, an aliquot of 7 µl was used for electrophoresis in a 0.8% agarose gel and examined as per section 22.214.171.124.
Kiwifruit ds cDNA purification
CHROMA SPIN TE-400 columns (Clontech, U.S.A) were used for the purification of ds cDNA. The columns were prepared by inversion until the gel matrix in the column became homogenous. Both top and bottom column caps were removed and the column placed in a 2 ml collection tube. The columns were centrifuged at 700g for 5 minutes in a microcentrifuge to remove the equilibration buffer and the collection tube discarded with the flow through. ds cDNA (93 µl) was introduced into the prepared CHROMA SPIN TE-400 column along with a new collection tube and centrifuged for 5 minutes at 700g to collect purified ds cDNA. Two purified kiwifruit ds cDNA samples were combined in a 1.5 ml microcentrifuge tube, likewise, the purified ds cDNA of mouse liver was taken in a separate 1.5 ml microcentrifuge tube. Ethanol precipitation of ds cDNA was carried out by incorporating 1/10th volume of 3 M sodium acetate, 2.5 volume of 100% ice-cold ethanol. The content was briefly mixed and incubated for one hour at -20°C. Then, microcentrifugation was carried out at 18,407g for 20 minutes at room temperature and the supernatant was removed without disturbing the pellet. Further centrifugation was carried out to empty out the remaining supernatant. Finally, the pellet was air-dried in a laminar airflow cabinet for 15 minutes, and 20 µl ultra-pure water was added to resuspend the ds cDNA.
Construction of kiwifruit Y2H cDNA library
Preparation of competent prey yeast cells
Yeast competent cell preparation and cDNA library generation were performed as described in YeastmakarTM yeast transformation system 2 user manual (Clontech, U.S.A). A freshly prepared YPDA (Yeast extract, Peptone, Dextrose and Adenine) agar plate was used to streak a frozen stock of Y187 prey cells. It was then sealed with Parafilm® M (Bemis Flexible Packaging, Neenah, WI, U.S.A) and incubated upside down at 30°C for 3 days. From the plate, one colony was chosen with a size range from 2 to 3 mm to inoculate 3 ml YPDA liquid medium in a sterile 15 ml Falcon® tube (Corning brand, U.S.A). The tube was incubated for eight and half hours at 30°C and 200 rpm in an orbital incubator (Gallenkamp; UK). Five µl of the culture was taken and introduced into a 250 ml sterile conical flask containing 50 ml YPDA liquid medium and incubated for 17 hours at 200 rpm to get an OD600 value of 0.2. The culture was transferred to a sterile 50 ml Falcon® tube (Corning brand, U.S.A) and centrifuged at 1,026g in a Sorvall® RC-5C Plus centrifuge (DuPont Instruments; Newtown, CT, U.S.A) for 5 minutes at room temperature. The supernatant was discarded, and the yeast pellet was resuspended in 100 ml fresh YPDA liquid medium, incubated for 3.10 hours at 30°C to reach OD600 0.5. The cultures were then split into two sterile 50 ml Falcon® tubes and centrifuged for 5 minutes at 1,026g and room temperature. The supernatant was poured off, and the pellet resuspended in 30 ml sterile deionized water and the centrifugation step was repeated. LiAc/TE (1:1) solution 1.5 ml (v/v) was used to resuspend the pellet which was later transferred into sterile 1.5 ml microcentrifuge tubes and microcentrifuged at 3,968g for 15 seconds. The supernatant was removed, and finally, yeast cells were resuspended in 600 µl of LiAc/TE (1:1) solution.
Yeast co-transformation of kiwifruit ds cDNA and library prey vector
In a sterile, pre-chilled 1.5 ml microcentrifuge tube the following were added: 20 µl ds cDNA, 6 µl pGADT7-Rec prey vector (500 ng/µl; Clontech, U.S.A) and 20 µl denatured Yeastmaker carrier DNA (10 µg/µl). They were gently mixed by pipetting up and down, after which 600 µl pre-chilled freshly prepared prey competent yeast cells were added and mixed by gentle pipetting. The mixture was transferred to a pre-chilled, and sterile 50 ml Falcon® tube, to which 2.5 ml freshly made 40% PEG/LiAc (Polyethylene glycol 3350/Lithium acetate) solution was added, mixed thoroughly and incubated for 45 minutes at 30°C with 15 minutes interval between regular hand mixing. This was followed by the addition of 160 µl DMSO (Invitrogen, U.S.A) and brief mixing. Yeast cells were heat shocked by placing the 50 ml Falcon® tube at 42°C for 20 minutes. The content was centrifuged at 1,026g for 5 minutes in a Sorvall® RC-5C Plus centrifuge to pellet yeast cells. The supernatant was removed, 3 ml YPD Plus medium (Clontech, U.S.A) was used to resuspend the pellet, which was then incubated at 30°C for 1.30 hours at 200 rpm in an orbital incubator. The centrifugation step was repeated to collect yeast cells, and the supernatant was discarded. Yeast cells were resuspended by adding 15 ml of 0.9% (w/v) sodium chloride.
Plating transformed yeast cells
Prey transformants that were resuspended in 0.9% (w/v) sodium chloride were serially diluted. Each dilution (100 µl) was plated on a Petri plate (150 mm x 20 mm) containing SD/-Leu agar medium. The remaining undiluted prey transformants were plated on 28 culture dishes of 500 cm2 size, (Corning brand, U.S.A) for each plate 450 µl transformants were plated and incubated at 30°C for 5 days. Yeast transformation efficiency and the total number of independent clones in the library were calculated according to the manufacturer’s protocol (Clontech, U.S.A).
Harvesting and collection of yeast transformants
After 5 days incubation, all SD/-Leu agar plates with yeast transformants were incubated at 4°C for 4 hours. Freezing medium (15 ml) (YPDA + 25% Glycerol) was added to each 500 cm2 culture dish and 5 ml freezing medium was added to each 150 mm x 20 mm Petri plate. Yeast colonies were removed from all plates with a sterile glass spreader. In a sterile 500 ml Schott Duran® bottle (DURAN Group; Wertheim, Germany) all yeast transformants were collected, and the cell density of the cDNA library was determined by hemocytometer using the standard formula below.
Average number of cells per 0.1 mm square x Dilution factor x 104
After the cell density assessment, many 1 ml cDNA libraries were aliquoted in 1.5 ml microcentrifuge tubes and snap frozen in liquid nitrogen, and placed at -80°C for long-term use.
Determining quality parameters of kiwifruit cDNA library
Plasmid isolation from yeast prey clones
Twelve cDNA prey yeast clones were randomly selected from the SD/-Leu agar medium for plasmid extraction. The plasmids were isolated by the combination of initial freezing and thawing steps and the ZyppyTM plasmid miniprep kit (Zymo Research, U.S.A). In a 1.5 ml microcentrifuge tube 500 µl MQ water was taken to which half a volume of prey clones was added. The tube was frozen at -80°C for 10 minutes, then thawed at room temperature followed by vortexing the tube for 5 minutes in a vortexer (Lab dancer; IKA®, Germany). This step was repeated once, after which, the sample was incubated at 65°C for 10 minutes and cooled briefly on ice. The sample was once more frozen at -80°C for 10 minutes and thawed. Hereafter, the ZyppyTM plasmid miniprep protocol was used. Lysis buffer (7x; 100 µl) was introduced into the tube containing freeze-thawed yeast clones, and the content mixed by inverting 6-8 times followed by incubation for 12 minutes at room temperature. Cold neutralization buffer (350 µl) was added and mixed thoroughly until the solution became homogeneous; the content was microcentrifuged at 13,523g for 5 minutes in a microcentrifuge. The supernatant of around 900 µl was taken and transferred into a column (Zymo-Spin™ IIN, Zymo Research, U.S.A) placed in a collection tube and microcentrifuged for 15 seconds at 13,523g. The flow-through was decanted off, the column was washed twice with 200 µl endo-wash buffer, 400 µl wash buffer for 30 seconds and two minutes respectively at 13,523g. Finally, the column was placed in a new 1.5 ml microcentrifuge tube, 15-20 µl ultra-pure water was added to the column and the elution was carried out by microcentrifugation at 13,523g for one minute.
E. coli transformation
For electroporation, 5 µl prey plasmid DNA and 8 µl one shot® electrocompTM E. coli cells (Invitrogen, U.S.A) were used. Both were added to a pre-cooled 1.5 ml microcentrifuge tube and mixed gently. The mixture was transferred into a 0.1 cm cuvette (Bio-Rad; Hercules, California, U.S.A) ensuring the mixture touched both sides of the cuvette by gentle tapping. BioRad GenePulser® II electroporator (Bio-Rad; Hercules, California, U.S.A) was utilized for electroporation using 1800 kV, 200 Ω (Resistance) and 25 µF (Capacity) parameters. Upon electroporation, 1 ml LB broth (Invitrogen, U.S.A) was used to resuspend the cells, which were then collected in a 1.5 ml microcentrifuge tube and incubated for one hour at 200 rpm and 37°C in an orbital incubator. After the incubation, E. coli cells were plated on LB agar containing ampicillin (100 µg/ml) and incubated overnight at 37°C. The transformants that emerged after electroporation were cultured overnight at 200 rpm and 37°C. The plasmid extraction was performed using the ZyppyTM plasmid miniprep kit (Zymo Research, U.S.A). The extracted plasmids were verified for the presence of cDNA insert by HindIII restriction digestion and fragmented on a 1% agarose gel as per section 126.96.36.199. These plasmids were sequenced (Macrogen, South Korea) and their cDNA inserts identified through nucleotide BLAST® query against the A. chinensis hybrid gene models and de novo transcript genome database (Plant & Food Research) and A. chinensis HY CK51F3_01 hybrid gene models. These two kiwifruit genome databases are generated from kiwifruit cDNA.
Kiwifruit total RNA purity analysis
Total RNA was isolated using the pine tree or CTAB method (Chang et al., 1993). Extractions were performed in 14 replicates of A. chinensis leaf sample because RNA isolation from kiwifruit is challenging due to the abundant presence of polysaccharide and polyphenol. The extracted total RNA purity was determined in a spectrophotometer by evaluating A260/280 and A260/230 ratios. As a result kiwifruit total RNA samples showed the A260/280 ratio ranging from 1.89-2.13, and the A260/230 ratio of 2.04-2.65. The absorbance values obtained for kiwifruit total RNA samples indicated the occurrence of no contaminants.
Kiwifruit total RNA integrity analysis
Kiwifruit total RNA integrity examination was carried out in a Bioanalyzer 2100. The Bioanalyzer methodology is robust and able to determine RNA concentration, length and distribution simultaneously using fluorimetry, microfluidics and capillary electrophoresis platforms respectively. Based on RNA length and distribution, RNA integrity number (RIN) values were allocated 1 to 10 (Schroeder et al., 2006). Kiwifruit total RNA samples evaluated for the integrity displayed RIN values from 2.30 to 8.10 (Figure 3.3). The capillary gel electrophoresis of RNA samples in the Bioanalyzer 2100 showed distinct 25S and 18S rRNA bands. In spite of that, the distribution of chloroplast RNA at regular intervals with different sizes were observed throughout total RNA (Figure 3.3). The Bioanalyzer 2100 which used relies on the integrity of mammalian 28S and 18S rRNA subunits to calculate a RIN. Whereas, plant total RNA has 25S and 18S rRNA units and ubiquitous chloroplast RNA. For this reason, the combination of RIN values and visual assessment of electropherogram are desirable to corroborate the integrity of plant RNA (Die and Roman, 2012). In this experiment, the visual inspection of the electropherogram was performed (Figure 3.4); consequently, clear 25S and 18S rRNA peaks were observed in RNA samples 2, 3, 5, 8, 9, 11, 12 along with small peaks of chloroplast RNA. The observed rRNA and chloroplast RNA peaks in the electropherogram were equivalent to the size of the bands noted in the capillary gel electrophoresis. Further, it showed no indication of decline in 25S and 18S rRNA signal intensities. Thereby, the kiwifruit total RNA was intact, and total RNA samples that acquired RIN values greater than 7 were used in the subsequent experiment.
Table of Contents
Table of contents
List of tables
List of figures
Chapter 1: General introduction
1.2 Pseudomonas syringae pv. actinidiae .
1.3 Plant-pathogen interaction
1.4 Plant recognition of pathogen effectors
1.5 Type III secretion system effectors of P. syringae
1.6 AvrPto family effectors of P. syringae
1.7 HopF family effectors of P. syringae
1.8 Host targets among P. syringae T3SS effector family
1.9 Importance of intrinsically disordered residues in bacterial efffectors
1.10 Psa and kiwifruit interaction
1.11 Research aims
Chapter 2: Finding PsaNZV-13 AvrPto5 host target in kiwifruit by a candidate host gene approach
2.2 Materials and Methods
Chapter 3: Construction and characterization of a kiwifruit in vivo Y2H cDNA library
3.2 Materials and Methods
Chapter 4: PsaNZV-13 AvrPto5 host target identification in kiwifruit by Y2H screening
4.2 Materials and Methods
Chapter 5: Functional analysis of PsaNZV-13 HopF2
5.2 Materials and Methods
Chapter 6: Concluding discussion
6.1 PsaNZV-13 AvrPto5 appears not to be a functional orthologue of PtoDC3000 AvrPto1
6.2 Y2H as a platform to identify plant pathogen-host targets
6.3 PsaNZV-13 AvrPto5 and AcHIPP26 interaction in Y2H and in planta
6.4 PsaNZV-13 AvrPto5 and HopF2 effectors are intrinsically disordered
6.5 PsaNZV-13 HopF2 does not associate with RIN4s in Y2H
6.6 Putative ADP-RT residues of PsaNZV-13 HopF2 are crucial for activity
6.7 Myristoylation motif may not be required for PsaNZV-13 HopF2 function in plants
6.8 NtRIN4 may be essential for PsaNZV-13 HopF2 mediated HR in N. tabacum .
6.9 Future work
6.10 Concluding comments .
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Functional characterization of Pseudomonas syringae pv. actinidiae effectors AvrPto5 and HopF2