Functional Genomic Analysis of Cotton Genes with Agrobacterium-Mediated Virus-Induced Gene Silencing

Abstract

Cotton (Gossypium spp.) is one of the most agronomically important crops worldwide for its unique textile fiber production and serving as food and feed stock. Molecular breeding and genetic engineering of useful genes into cotton have emerged as advanced approaches to improve cotton yield, fiber quality, and resistance to various stresses. However, the understanding of gene functions and regulations in cotton is largely hindered by the limited molecular and biochemical tools. Here, we describe the method of an Agrobacterium infiltration-based virus-induced gene silencing (VIGS) assay to transiently silence endogenous genes in cotton at 2-week-old seedling stage. The genes of interest could be readily silenced with a consistently high efficiency. To monitor gene silencing efficiency, we have cloned cotton GrCla1 from G. raimondii, a homolog gene of Arabidopsis Cloroplastos alterados 1 (AtCla1) involved in chloroplast development, and inserted into a tobacco rattle virus (TRV) binary vector pYL156. Silencing of GrCla1 results in albino phenotype on the newly emerging leaves, serving as a visual marker for silencing efficiency. To further explore the possibility of using VIGS assay to reveal the essential genes mediating disease resistance to Verticillium dahliae, a fungal pathogen causing severe Verticillium wilt in cotton, we developed a seedling infection assay to inoculate cotton seedlings when the genes of interest are silenced by VIGS. The method we describe here could be further explored for functional genomic analysis of cotton genes involved in development and various biotic and abiotic stresses.

1. Introduction

Cotton (Gossypium spp.) is widely planted around the world for its significant economic value of the textile fiber, feed, foodstuff, oil, and biofuel products (1). The upland cotton, G. hirsutum, dominates world cotton commerce with more than 90% of the annual fiber production (2, 3). As of 2009/2010, cotton cultivation was estimated to be about 30 million hectares worldwide, producing a value of approximately 39 billion US dollars, while its production in the United States alone was estimated at approximately five billion dollars (4).

The major concern for cotton production is the significant loss of yield caused by many devastating diseases and pests. Cotton has been considered as the world's dirtiest crop due to the heavy application of pesticides and fungicides. The soilborne pathogen Verticillium dahliae causes severe Verticillium wilt diseases on cotton (5). Because of extremely persistent resting structures, such as microsclerotia, this pathogen can survive in soil for many years. Most notably, this fungus is very difficult to be reached by fungicides because the fungi reside in the woody vascular tissues and can be transmitted systemically in cotton plants (5). Despite the significant efforts towards understanding the biology of this pathogen and identifying the genetic determinants responsible for cotton Verticillium resistance (6, 7), to date, the genetic and molecular mechanisms underlying cotton resistance to Verticillium infection remain poorly understood.

In recent years, significant advances in cotton genetics and genomics have been achieved towards the molecular breeding and genetic engineering of new cotton varieties to increase the sustainable yield and fiber quality as well as to improve the traits combating various pathogen infections (8, 9). Understanding cotton gene functions and regulations constitutes a critical step for manipulating cotton genes in agriculture. A persistent challenge in cotton functional genomic studies is the lack of molecular and genetic tools partly due to the large genome size, the long growth cycle, and the unstable transformation efficiency (9). Virus-induced gene silencing (VIGS) has been demonstrated as a rapid and efficient approach to study gene functions at whole-genome level in various plant species (10–12). VIGS, a type of RNA-mediated posttran-scriptional gene silencing, functions as an antivirus defense mechanism in plants (10–12). Through Agrobacterium infiltration, the T-DNA containing the partial viral genome and gene of interest is delivered into host cells. The production of double-stranded RNAs between the endogenous gene and DNA fragment delivered from T-DNA vector results in a chain reaction to generate robust silencing signals (12). With the time, the silencing of endogenous genes occurs both locally and systemically throughout the plant tissues.

To date, different plant virus vectors have been deployed for VIGS assays in dicotyledonous plant species, including tobacco mosaic virus (TMV), potato virus X, tomato golden mosaic virus, tobacco rattle virus (TRV), and cotton leaf crumple virus (CLCrV) vectors (13–16). In monocotyledonous plants, barley stripe mosaic virus has been applied to silence genes in barley and wheat and brome mosaic virus (BMV) in rice (17–19). Among these viruses, TRV invades a wide range of hosts and spreads vigorously throughout the entire plants but usually triggers a mild symptom, which makes it a good candidate as a VIGS vector (13). TRV belongs to the Tobravirus containing a bipartite positive-sense single-stranded RNA: RNA1 and RNA2. RNA1 contains genes of the viral replicase, RNA-dependent RNA polymerase, and movement protein, which are required for replication and movement (13). RNA2 contains genes encoding the coat protein and other nonessential structural proteins, which can be engineered to insert a target gene fragment to be silenced. Both RNA1 and RNA2 cDNAs have been cloned into T-DNA cassette between duplicated 35S promoter and the nopaline synthase (NOS) terminator (13). Here we describe a detailed method of Agrobacterium infiltration-based VIGS assay in cotton seedlings. We further provide an example of using VIGS assay to understand gene functions in cotton seedling resistance to Verticillium dahliae infection. The protocol established here could be potentially adapted to study a diverse array of biotic and abiotic stress responses in cotton and provides a powerful tool in cotton functional genomics.

2. Materials

2.1. Plants, Growth Conditions, and Pathogen Strain

1. Cotton seeds: upland cotton (Gossypium hirsutum) variety FM9160 seeds obtained from Bayer CropScience (Lubbock, TX, USA).

2. Soil: Metro Mix 700 (SunGR, Beavile, WA, USA).

3. Growth room conditions: 23–25°C and 120 μE m⁻² s⁻¹ light, with a 12 h light/12 h dark photoperiod.

4. Pathogen strain: Verticillium dahliae (isolate King).

2.2. Plasmid Construction and Cloning

1. PCR amplification reagents: 10× reaction buffer, 10 mM dNTP, and Phusion high-fidelity DNA polymerase (New England BioLabs, MA, USA).

2. Restriction enzymes: EcoRI and KpnI (New England BioLabs, MA, USA).

3. DNA ligation kit: 10× T4 DNA ligase buffer and T4 DNA ligase (4 U/μl) (New England BioLabs, MA, USA).

4. VIGS RNA2 vector: pYL156 (pTRV2:RNA2).

5. QIAquick Gel Extraction Kit (QIAGEN).

6. LB liquid medium.

7. LB plates containing antibiotics.

8. Kanamycin (50 mg/ml stock) and gentamicin (50 mg/ml stock).

9. Agrobacterium tumefaciens GV3101 electro-competent cells stored in 10% glycerol at −80°C.

2.3. Agrobacterium Infiltration for VIGS Assay and Confirmation of Gene Silencing

1. Agrobacterium tumefaciens GV3101 containing pTRV1 (pTRV-RNA1).

2. Agrobacterium induction culture solution: LB liquid medium containing 50 μg/ml of kanamycin, 50 μg/ml of gentamicin, 10 mM of MES (2-(4 morpholino)-ethane sulfonic acid), and 20 μM acetosyringone.

3. Agrobacterium infiltration solution: 10 mM MgCl₂ containing 10 mM of MES and 200 μM acetosyringone.

4. 1 ml needleless syringes.

5. Syringe needles (20 Gauze).

6. Spectrum™ Plant Total RNA Kit (Sigma).

7. cDNA synthesis kit (Invitrogen).

8. PCR machine.

3. Methods

3.1. Grow Cotton Plants

1. Fill the soil in square pots (7 cm in diameter) and put the pots in a tray.

2. Sow the seeds in the soil (one seed per pot).

3. Soak the potting soil by pouring water in the tray.

4. Cover the tray with a plastic dome; grow the seedlings in the growth room until two cotyledons have emerged and remove the dome. Growth room condition: 23–25°C and 120 μE m⁻² s⁻¹ light with a 12 h light/12 h dark photoperiod (see Note 1).

3.2. Clone Cla1 and Other Genes of Interest into pYL156 Vector

1. Search your genes of interest through blast against the Gossypium unigenes database at http://www.cottondb.org/blast/blast.html. Design a pair of primers that could amplify about 500 bp of target genes with EcoRI at 5′ end and KpnI at 3′ end. We used Arabidopsis Cloroplastos alterados 1 (AtCla1, AT4G15560) as a query for the blast search for cotton Cla1 gene (20) (see Notes 2 and 3).

2. A 500 bp fragment of Cla1 gene or other genes of interest could be amplified by PCR with Phusion high-fidelity DNA polymerase from the cDNA library synthesized with RNA isolated from cotton leaf tissues (see Notes 4 and 5).

3. PCR products are purified with ethanol precipitation and digested with EcoRI and KpnI, together with pYL156 vector. The digestion is done in 15 μl mixture containing 0.2 μl of each enzyme, 1.5 μl of NEBuffer 4, and 0.15 μl of 100× BSA. The digestion is conducted at 37°C for 2–3 h.

4. Recover the digested vector and PCR fragments from DNA agarose gel using QIAquick Gel Recover Kit following the manufacturer's instruction.

5. Ligate the digested PCR products with vector: the ligation is done in a 15 μl mixture containing 1 μl of vector DNA (5–10 ng), 5 μl of insert DNA (20–50 ng), 1.5 μl of 10× T4 DNA ligase buffer, and 0.2 μl of T4 DNA ligase (4 U/μl). The reaction is incubated at room temperature for ∼2 h.

6. Add 50 μl of E. coli competent cell into the ligation mixture, heat shock at 37°C for 2 min; chill on ice; add 200 μl LB liquid; and recover at 37°C for 30 min with a roller drum.

7. Spread the E. coli culture on LB plate containing 50 μg/ml kanamycin, and incubate the plate at 37°C overnight.

8. Screen the clones by miniprep DNA isolation and digestion of the DNA with EcoRI and KpnI. Finally, sequence the clones to confirm the insertion.

9. The confirmed clones are transformed into Agrobacterium tumefaciens GV3101 by electroporation method.

10. Spread the culture on LB plate containing kanamycin (50 μg/ml) and gentamicin (50 μg/ml); incubate at 28°C for 2 days. Culture the Agrobacteria with LB liquid, and store the culture with 25% glycerol at −80°C for further use.

3.3. VIGS Processes

1. Select a single colony from the fresh LB plates containing Agrobacterium tumefaciens carrying pTRV1, pYL156 (empty vector control), pYL156-GrCla1, and pYL156 carrying Your Favorite Gene (pYL156-YFG).

2. Inoculate the single colony with 5 ml of LB liquid containing kanamycin (50 μg/ml) and gentamicin (50 μg/ml); culture at 28°C overnight on a roller drum at 50 rpm.

3. Add 45 ml of Agrobacterium induction culture solution into the above culture; incubate at 28°C overnight in a shaker at 100 rpm.

4. Harvest the bacteria at 1,180 × g for 5 min, resuspend the pellet in Agrobacterium infiltration solution, and adjust the OD 600 to 1.5.

5. Leave the bacterial cultures on the bench at room temperature for 3 h (see Note 6).

6. Gently punch a couple of holes on the backside of the cotton cotyledons using a fine-tip needle without piercing through the tissue (see Note 7).

7. Mix the Agrobacterial culture suspension of pTRV1 with pYL156, pYL156-GrCla1, or pYL156-YFG at a 1:1 ratio.

8. Hand infiltrate the mixture into the cotyledons through the wounding sites using a needleless syringe.

9. Cover the infiltrated plants with a plastic dome and leave at room temperature overnight under the dimlight (30 μE m⁻² s⁻¹) (see Note 8).

10. Transfer the plants to the growth room and remove the dome.

11. About 7–10 days later, examine the silencing phenotype for Cla1 or YFG. The true leaves on the plants infiltrated with pYL156-GrCla1 will show albino phenotype (see Note 9).

12. Harvest the true leaves from silenced and control plants; isolate RNA with Spectrum™ Plant Total RNA Kit (Sigma).

13. Synthesize cDNA with cDNA synthesis kit (Invitrogen), and perform semiquantitative or real-time RT-PCR analysis to confirm the silencing of endogenous genes (see Note 10).

3.4. Verticillium Infection of VIGS Silenced Cotton Plants

1. Culture Verticillium dahliae (isolate King) on potato dextrose agar (PDA) plates at room temperature (23°C) for 3–4 days.

2. When pYL156- GrCla1-silenced plants show albino symptoms, prepare the spore suspension of V. dahliae.

3. Collect the fungal spores by scratching the fungal mycelium on the plate surface using sterile dH₂O. The spores are filtered with autoclaved cheesecloth, counted under a microscope using a hemacytometer, and diluted to the concentration of 1 × 10⁶ /ml in sterile H₂O containing 0.001% Tween 20.

4. Infiltrate 100 μl of spore suspension to the stems of silenced cotton plants at a position approximately 1 cm below cotyledons using a 22 Gauze needle syringe (see Note 11).

5. Score the Verticillium wilting phenotype by examining the wilting symptoms appeared on the true leaves of plants (see Notes 12–14).