A Functional Genomics Method for Assaying Gene Function in ...

Chapter 13 A Functional Genomics Method for Assaying Gene Function in Phytopathogenic Fungi Through Host-Induced Gene Silencing Mediated by Agroinfiltration Vinay Panwar, Brent McCallum, and Guus Bakkeren Abstract With the rapid growth of genomic information, there is an increasing demand for efficient analysis tools to study the function of predicted genes coded in genomes. Agroinfiltration, the delivery of gene constructs into plant cells by Agrobacterium tumefaciens infiltrated into leaves, is one such versatile, simple, and rapid technique that is increasingly used for transient gene expression assay in plants. In this chapter, we focus on the use of agroinfiltration as a functional genomics research tool in molecular plant pathology. Specifically, we describe in detail its use in expressing phytopathogenic fungal gene sequences in a host plant to induce RNA silencing of corresponding genes inside the pathogen, a method which has been termed host-induced gene silencing (HIGS). We target the fungal pathogen Puccinia triticina which causes leaf rust on its wheat host, but the method is applicable to a variety of pathosystems. Key words Agroinfiltration, Transient expression, Plant–fungal interactions, Rust fungi, RNA interference, Gene silencing, HIGS

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Introduction The management of fungal plant pathogens is of utmost importance to curb losses in agriculture production systems and feed the increasing global population. Factors influencing the interaction of pathogenic fungi with their hosts have been a major research topic in the fungal research community in recent years. In spite of major efforts to develop new fungicides and resistant plant varieties, losses due to fungal diseases, especially in agronomically important crop plants, are a growing stimulus for basic research in this field. One viable alternative is to identify pathogenicity determinants essential for disease development which can then be specifically targeted. In recent years, a number of molecular genetic tools for the identification and functional analysis of genes involved in the interplay of pathogenic fungi and their host plants have been developed [1–5]. However, there is a paucity of effective

Kirankumar S. Mysore and Muthappa Senthil-Kumar (eds.), Plant Gene Silencing: Methods and Protocols, Methods in Molecular Biology, vol. 1287, DOI 10.1007/978-1-4939-2453-0_13, © Springer Science+Business Media New York 2015

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functional genetic tools for biotrophic (nonculturable) fungi due to difficulty in their genetic transformation [6]. RNA interference (RNAi) or gene silencing, a widely used technology for functional gene analysis [7–9], has become an emerging strategy for the study of genes in fungi as well. Because of the lack of transformation techniques for biotrophic fungi, alternative strategies based on gene silencing can be effectively used to study and analyze gene function in these organisms. A “proof-ofconcept” study showed the feasibility of using RNAi-based “hostplant-mediated pathogen gene silencing” in the powdery mildew fungus Blumeria graminis-barley pathosystem [10]. This strategy, which was termed Host-Induced Gene Silencing or HIGS, depends on the uptake by the invading pathogen of small interfering RNA (siRNA) molecules generated in the host plant but specific to endogenous fungal genes. When targeting genes vital for pathogen infection or disease development (true pathogenicity genes), fungal development, and disease can be suppressed. To induce silencing, delivery and expression of silencing constructs in cereal host plants can be achieved by the Barley stripe mosaic virus (BSMV). This virus-induced gene silencing (VIGS) strategy was shown to downregulate the expression of endogenous genes in the barley powdery mildew fungus Blumeria graminis [10], and the function of several virulence effector genes was revealed this way [11]. The VIGS strategy using BSMV was successfully applied to wheat-infecting biotrophic fungi of the genus Puccinia, also resulting in downregulation of the expression of endogenous rust fungus genes [12–14]. For the cereal-rust pathosystem, we also successfully explored the use of an Agrobacterium- mediated infiltration (agroinfiltration) assay [15]. By choosing predicted pathogenicity genes, we showed the generation of rust fungal gene-specific siRNA molecules in host wheat plants that subsequently induced silencing of the corresponding target genes inside the pathogen; ensuing disease suppression demonstrated their role in pathogenicity [14, 15]. Very recently, HIGS induced from integrated constructs in transgenic barley was shown to suppress disease development by a different fungus, Fusarium graminearum [16]. Agroinfiltration is a widely used technique in which the bacterium Agrobacterium tumefaciens is exploited to mediate transient expression of desired genes in plants [17, 18]. A. tumefaciens possesses a natural gene transfer mechanism by which a DNA segment (T-DNA) from its tumor-inducing plasmid (pTi) is transferred to plant cells where it can be expressed. The T-DNA can be engineered as an efficient plant transformation system [19]. In the agroinfiltration method, a suspension of A. tumefaciens carrying the silencing construct within its T-DNA region behind a strong plant-specific promoter is injected into an intact plant leaf. Once inside the plant leaf, the bacterium transfers the silencing construct as T-DNA to plant cells which can either integrate into the host genome or remain present in an extra-chromosomal form, both of which can

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be transcribed. Agroinfiltration results in strong transient expression due to a high copy number of nonintegrated T-DNA [20] and can also trigger an RNAi response due to a high level of gene expression [21]. The infiltrated plant can be monitored for a possible effect on phenotype, subjected to experimental conditions, or harvested and used for molecular analysis. The benefit of agroinfiltration compared to stable plant transformation is speed and convenience, given that generation of transgenic plant lines is costly and labor intensive. However, although transient assays are convenient for initial testing when successful, expressing constructs often need to be used to yield genetically stable plant lines for further testing. Agroinfiltration has been exploited as an efficient and versatile tool for studying foreign gene expression [22], gene silencing [23, 24], host–pathogen interactions [25, 26], protein–protein interactions [27, 28], protein production [29, 30], and signal transduction pathways [31], to name a few. For transient assays, A. tumefaciens is usually applied using vacuum infiltration or by syringe infiltration. Vacuum infiltration has the advantage that large areas, even entire plants, can be infiltrated at once, whereas infiltration by syringe, although easy to perform and more targeted, is usually applied on a smaller scale. Agroinfiltration by syringe has been applied successfully in a variety of plant species, including Nicotiana spp., tomato, lettuce, Arabidopsis, flax, pea, grapevine, pepper, and rose. In recent years, agroinfiltration has become a key reverse genetics research tool in RNAi studies. The main limitation of agroinfiltration is that, although it works very efficiently for a number of dicotyledonous plants, it is not very successful in many other species, especially monocots, which are recalcitrant to A. tumefaciens transformation [32]. Nevertheless, several monocot species have been shown to be susceptible to genetic transformation by A. tumefaciens [33–35]. Therefore, it is feasible to develop an efficient Agrobacterium-mediated transient expression system using highthroughput RNAi strategies [36] although the technology has not been efficiently tested and applied in monocots. Recently, successful Agrobacterium-mediated transient gene expression has been reported for several monocotyledonous species, including switchgrass [37], rice [38, 39], and anthurium [40]. In this chapter, we describe in detail a generic method for host-induced gene silencing of rust fungal genes in wheat plants using agroinfiltration.

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Materials Plant Growth

1. Seeds of Triticum aestivum (wheat) (see Note 1). 2. Square Dura Pots (3.5″), germ trays, and clear domes (that fit the germ trays). 3. Standard germination soil (substrate no. 1) and potting soil (no. 3) for plant growth.

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1. For the agroinfiltration assay described in this chapter, we have used the wheat leaf rust fungus Puccinia triticina (Pt) MAP Kinase (PtMAPK) gene fragment (see Note 2) cloned in hairpin RNA (hpRNA) conformation in the cereal-specific binary expression vector pIPKb007 (Fig. 1; ref. 41).

2.2 Binary Vectors and Agrobacterium Strains

2. A. tumefaciens strain COR308 harboring pIPKb007 RNAi vector (see Note 3). Supervirulent A. tumefaciens strain pIPKb007_PtMAPK R1

PtMAPK R2 I

ColE1

PtMAPK R2

Ubi1pro

pVS1

R1

RB

T

r

T Hpt

Ubi1pro

LB

Specr pIPKb007 -PtMAPK

AgtCOR308

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RNAi vector construction for HIGS

and A. tumefaciens transformation

Phenotypic and molecular analysis of HIGS effect on disease progression

Preparation of Agrobacterium culture

Syringe infiltration

Challenge by fungal pathogen

Fig. 1 A schematic representation of the agroinfiltration process used for the HIGS assay. Top line: T-DNA delineated by the right border (RB) and left border (LB) elements, and having P. triticina PtMAPK gene fragments cloned in inverted orientation so as to result in a hairpin RNA form when expressed from the maize ubiquitin1 promoter (Ubi1pro). R1 and R2 refer to the GateWay-specific recombination sites; the hygromycin resistance cassette (Hptr) is not used in this assay. The T-DNA resides in cereal-specific binary silencing vector pIPKb007 with broad host range origins of replication and spectinomycin (Specr) resistance [41]. The Agrobacterium culture with the binary vector having the T-DNA construct (red fragment) is prepared and infiltrated by syringe into the first fully developed leaf of a two- to three-leaf-stage wheat plant. 2–3 days postagroinfiltration, the plant is challenged with fungal urediniospores. The infiltrated leaf section is observed for disease response. The photograph shows wheat plants agroinfiltrated with the PtMAPK silencing construct, resulting in suppression of rust disease symptoms in the infiltrated area 10 days after challenge with urediniospores, as compared to Agrobacterium alone (Agt-COR308) and buffer control (BC)


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COR308 is a recA-deficient C58 nopaline strain UIA143 harboring disarmed pTi derivative plasmid pMP90 and a special vir helper plasmid pCH32, which provides extra copies of the virA and virG two-component signaling genes. It can be obtained from Cornell University (http://www.biotech. cornell.edu/BIBAC/BIBACHomePage.html). 2.3 Media, Buffers, and Solutions

1. Luria-Bertani mannitol (LB) medium: Dissolve 10 g Bactotryptone, 5 g yeast extract, and 10 g NaCl in 950 mL deionized water. Adjust the pH of the medium to 7.0 using 1 N NaOH, and bring the volume up to 1 L. For preparing LB agar plates, add 15 g/L agar and autoclave on liquid cycle for 20 min at 15 psi. After autoclaving, cool to approximately 55 °C, add required antibiotics and pour into Petri dishes. Let harden, then invert the plates and store at 4 °C. 2. Yeast extract and beef (YEB) medium: Dissolve 1 g yeast extract, 5 g beef extract, 5 g peptone, 5 g sucrose, 0.493 g MgSO4.7H2O in 950 mL deionized water. Adjust the pH of the medium to 7.2, and make up the volume to 1 L. Autoclave the medium at liquid cycle for 20 min at 15 psi. Cool down at room temperature, and store at 4 °C. 3. Antibiotics: For A. tumefaciens stain COR308 carrying the PtMAPK RNAi construct, we used spectinomycin (100 mg/mL) and tetracyclin (5 mg/ mL) (see Note 4). 4. Acetosyringone: Stock of acetosyringone (3, 5-dimethoxy4hydroxyacetophenone) is prepared in filter-sterilized DMSO at a concentration of 0.5 M (see Note 5). Dispense in aliquots, and store at −20 °C. 5. 1 M MES (2-[N-morpholino] ethanesulphonic acid): dissolve 42.64 g MES in 200 mL H2O, and adjust pH to 5.6 with NaOH; filter-sterilize. 6. Infiltration medium: Prepare a final concentration of 10 mM MES, 10 mM MgCl2, and 200 µM acetosyringone in deionized water; filter-sterilize (see Notes 6 and 7).

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Methods

3.1 Plant Growth Conditions

1. Germinate four or five wheat seeds in 3.5″ square Dura pots containing standard germination soil at 25 °C with 16 h light and 8 h dark period with 74 µmol/m2 s light intensity and 55–65 % relative humidity. 2. 7 or 8 days after sowing, plants are at the optimal developmental stage to be used for agroinfiltration. At this stage, plants have at least one fully developed true leaf (see Note 8).

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3.2 Culturing and Preparation of A. tumefaciens Suspension

1. Inoculate 10 mL of YEB starter culture containing desired antibiotics with a single colony of A. tumefaciens strain harboring the desired plasmid. Grow the starter culture up to 24 h at 28 °C under constant agitation (200 rpm) to reach log phase (see Notes 9 and 10). 2. Inoculate 5 mL of starter culture into 30 mL of YEB medium containing antibiotics in the desired concentration, 10 mM MES and 20 µM acetosyringone (see Note 11). 3. Incubate the Agrobacterium culture in the dark at 28 °C with shaking (200 rpm) overnight (see Note 12). 4. Spin down the bacterial cells by centrifugation at 4,000 × g at 4 °C. 5. Carefully wash the pellet twice with ice-cold sdH2O without disturbing the pellet. Remove all supernatant and dispense the pellet in 15 mL (ice-cold) infiltration medium by tapping or gentle vortexing (see Note 13). 6. Pellet the bacterial cells by centrifugation at 4,000 × g at 4 °C. 7. Discard the supernatant without disturbing the bacterial pellet. Gently resuspend the cells in infiltration medium to the required final OD600 value (see Note 14). 8. After adjusting the OD600, keep the Agrobacterium suspension to be used for agroinfiltration at room temperature (22–25 °C) for approximately 3 h in the dark without agitation.

3.3 Leaf Selection, Agroinfiltration, and Plant Incubation

1. Select healthy plants for agroinfiltration assays (see Note 15). 2. Gently make a small scratch in the epidermis at the abaxial surface (lower side) of the wheat leaf using a needle (see Notes 16 and 17). 3. Fill a 1 mL needleless syringe with the Agrobacterium suspension. Hold the leaf to be infiltrated between gloved index finger and syringe; place the syringe tip against the scratched side of the leaf and infiltrate gently, simultaneously applying gentle counter pressure on the other side of the leaf with your finger (see Note 18). 4. The Agrobacterium suspension is slowly injected through tiny incisions made on the underside of the leaf by pushing the syringe piston slowly down with the thumb (see Notes 19–21). 5. After infiltration, keep plants at room temperature (22–25 °C) for 15 min to dry any excess Agrobacterium suspension from the surface of infiltrated leaves (see Note 22). 6. Place the pots with the infiltrated plants in a flat tray, and cover with a plastic dome (see Note 23). 7. Transfer plants to a growth chamber, and let them rest for approximately 2–3 days in the dark at 21–23 °C (see Notes 24 and 25).


3.4 Postinfiltration Assays and Fungal Inoculations

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1. For molecular studies, collect the leaf samples at approximately 72 h postinfiltration (see Notes 26 and 27). 2. For subsequent fungal challenge inoculations, expose agroinfiltrated plants to light approximately 48 h postinfiltration (see Note 28). 3. Observe plants for fungal disease development. For molecular assays, agroinfiltrated tissue can be harvested after challenging with the pathogen at different time points as desired by experiments.

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Notes 1. In Agrobacterium-mediated transformation, the genotype of the plant is generally found to influence T-DNA transfer and transgene expression. 2. Choice of the candidate pathogen gene to be targeted for RNAi is an important parameter if the HIGS assay is to result in disease suppression. HIGS might result in silencing of the targeted gene, but if the gene function is not essential for the pathogen there may be no effect observed on the disease phenotype [12]. 3. Several Agrobacterium strains are available for the transfer of binary plasmids carrying genes of interest. The choice of Agrobacterium strain is important because of specific host– bacteria interactions. It is always desirable to compare agroinfiltration efficiencies with at least two or three virulent Agrobacterium strains for a particular plant species. 4. For all other vectors, antibiotics can be prepared in appropriate solvents and added at the required concentrations. Stock solutions for antibiotics can be prepared and stored at -20 °C. 5. Use teflon or nylon membrane filters (0.2 µ) to filter-sterilize DMSO. 6. Protect the acetosyringone solution from light during use, and make fresh stock every 2–3 months. Add acetosyringone immediately before use. 7. Prepare fresh infiltration medium; protect from direct light. 8. Selection of the appropriate leaf for infiltration is very important for efficient transient expression. Among different treatments or plants, leaves should be at the same developmental stage for better comparison of results and minimizing variations in large-scale experiments. 9. Transformation of Agrobacterium with the desired plasmid can be performed as described by Annamalai and Rao [42]. Transformed colonies are selected on medium containing antibiotics, and the presence of the target gene in the transformants

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is confirmed by polymerase chain reaction (PCR) or restriction enzyme digestion. Positive clones can be stored at −80 °C in 30 % glycerol. To prepare the starter culture, an Agrobacterium colony is picked from a fresh culture plate (LB or YEB medium with 15 g/L agar). It is desirable to prepare fresh bacterial culture plates from the glycerol stock; check all clones by PCR, using gene-specific primers, each time a new plate is made prior to use in infiltration assays. Stop incubating the starter culture when OD600 has reached 1.0–1.5. 10. If possible, mix two or three individual colonies from different Agrobacterium clones transformed with the same construct to minimize the chance of any variability in expression of the target gene due to silent mutations or target gene sequence deletions that may occur during culturing. 11. Inducing Agrobacterium Vir genes with acetosyringone is important for an efficient assay. 12. Measure OD600 of the overnight culture, and stop incubating the culture when it reaches 0.8–1.5. 13. The washing step is done to remove all traces of growth media and antibiotics which otherwise might interfere with transformation efficiency. Do not dispense the bacterial pellet by vortexing too hard which can damage bacterial cells. 14. The optimal OD600 used for infiltration depends on the gene of interest and plant genotype. We recommend testing different ODs; typically, for the HIGS assay, we use an OD600 in the range of 0.5–1.0. In our agroinfiltration assay, we found an OD600 of 0.6–0.75 is optimal. At a higher OD, a higher concentration of bacteria may result in a hypersensitive reaction with necrosis and localized cell death along the infiltrated areas, whereas a lower OD may result in insufficient delivery of target genes into the plants with almost no detectable gene expression. 15. Generally, wheat leaves are difficult to infiltrate. We found that not watering plants 1 or 2 days before agroinfiltration improves the infiltration efficiency. But do not let plants get too dry. If the plant starts wilting, the additional shock of infiltration will be enough to kill the leaf and even the very young plant. Plantlets should look healthy. 16. The developmental and physiological stage of the plant affects the efficiency of the transient expression assays. The first fully developed leaves of 7- to 8-day-old wheat plantlets grown under greenhouse conditions are best for infiltration. We did not have much success with transient expression in older leaves. 17. Make sure not to scratch the leaf so hard as to pierce the leaf through both sides, as the bacterial suspension will pass through the puncture to the other side of the leaf.


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18. Wear glasses and a face shield to protect face and eyes from bacterial suspension squirting upwards during syringe infiltration. Gloves should also be worn during infiltration and changed with each different bacterial culture to avoid contamination. 19. Do not force entry of the Agrobacterium suspension too hard as this will damage the leaf cells with reduced transient expression efficiency and result in visible HR-like or necrotic symptoms. It is advisable to infiltrate the bacterial suspension to the point where it can easily migrate to the apoplastic spaces. Movement of bacterial suspension in the apoplastic spaces is clearly visible by the formation of dark green sections. Continue to inject the Agrobacterium suspension until the dark green section no longer expands. Multiple infiltration points may be necessary to infiltrate an entire leaf. 20. Compared to dicots, in cereals, the leaf morphology and structure of the leaf epidermis prevent infiltration of bacterial suspension by simple pressure. 21. Agroinfiltration is a delicate manipulation, and dexterity comes with experience. 22. Suck away any excess bacterial suspension from the surface of infiltrated leaves with a soft facial tissue. Do not wipe or rub the leaf. 23. It is advisable to use trays with drainage holes and domes with small holes for aeration. 24. Plants should postinfiltration.

remain

well

hydrated

and

nourished

25. Keep infiltrated plants in well-controlled growth chambers as plant RNAi is temperature dependent, and growth conditions can therefore affect the progression of gene expression. 26. In agroinfiltration assays, gene expression peaks approximately 3–4 days postinfiltration and declines thereafter. 27. It is advisable to use fresh leaves for all molecular assays. In wheat, we observed quite often that infiltrated leaves become transparent or waterlogged. After incubating plants in the dark postinfiltration, such leaves quickly develop necrotic symptoms and collapse when exposed to light. We generally do not include such leaves for any molecular analysis or HIGS assay. In wheat, we did not observe any systemic spread of silencing, and it was more or less observed within the infiltrated zone of the leaf. Therefore, tissue samples for molecular analysis are collected from within the infiltrated areas. 28. Before challenging with fungus, expose plants to light (under greenhouse conditions) for approximately nine to 10 h to recover and firm up. Inoculations with rust fungi require plants to be kept under highly humid conditions for nearly 12–16 h.

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