Functional Aspects of Silencing and Transient Expression of psbS in Nicotiana benthamiana

Richard B. Peterson, Hillar Eichelmann, Vello Oja, Agu Laisk, Eero Talts, Neil P. Schultes

Department of Biochemistry and Genetics, The Connecticut Agricultural Experiment Station, New Haven, USA.
Institute of Cell and Molecular Biology, Tartu University, Tartu, Estonia..

DOI: 10.4236/ajps.2013.47184                

Abstract

MicroRNA-based gene silencing is a functional genomics tool for a wide range of eukaryotes. As a basis for broader application of virus-induced gene silencing (VIGS) to photosynthesis research, we employed a tobacco rattle virus (TRV) vector to silence expression of the nuclear psbS gene in Nicotiana benthamiana. The 22-kiloDalton psbS protein is essential for xanthophyll- and H⁺-dependent thermal dissipation of excitation in higher plants widely known as nonphotochemical quenching (NPQ). Controls treated with the TRV-VIGS vector containing a bacterial chloramphenicol resistance gene as the silencing target were included to test for non-silencing effects of the viral vector system. PsbS protein was undetectable and both psbS mRNA transcript levels and NPQ capacity were dramatically reduced in new leaf tissue of VIGS-psbS plants only. Photosynthetic performance in TRV-VIGS-treated and uninfiltrated plants was assessed by application of CO₂ exchange, chlorophyll fluorescence, and in vivo absorbance changes at 810 nm. TRV-VIGS caused a mild stress based on pigment content and light absorption characteristics in some cases. To assess transient complementation of NPQ, the endogenous psbS gene was silenced using only the transit sequence in the TRV vector followed by Agrobacterium-mediated transient expression of a modified gene consisting of an altered transit sequence fused to the native mature protein sequence. Nevertheless, NPQ in infused fully expanded leaves that expressed this re-introduced form was not fully restored indicating the possible importance of psbS incorporation prior to formation of grana stacks.

Keywords

Fluorescence; Gas Exchange; Nonphotochemical Quenching; Quantum Yield; 810-nm Absorbance

1. Introduction

The exponential growth in gene sequence information has led to use of reverse genetics to dissect complex functions by selective protein elimination. Most efforts have relied on permanent modification of the host genome by mutation or transformation. However, transgenic complementation studies are beset by limitations imposed by transformation frequency and silencing. Screening for deletion alleles is laborious and appropriate T-DNA or transposon insertion “knock-out” lines may be unavailable or non-viable as homozygotes.

Virus-induced gene silencing (VIGS) in plants can substantially improve the speed and convenience of the reverse genetics approach [1,2]. Post-transcriptional silencing of endogenous genes [3,4], induced by engineering genomic sequences into RNA genome viruses [5,6], circumvents the need to generate stable transgenic or mutant stocks. Silencing does not rely upon integration of transgenes into host genomes. Virus propagation plus dissemination and microRNA amplification plus transport combine to deliver VIGS at high levels in growing parts. Suppression of gene expression is often far higher than is achieved in stable antisense transgenic lines [7]. Gene silencing is highly sequence specific, requiring only a short section of perfect nucleic acid identity. Selective silencing of individual highly similar gene family members can be accomplished by targeting less conserved 5’ or 3’ UTR or promoter sequences. Alternatively, gene families can be co-silenced if shared sequences are targeted.

Photosynthesis is a dynamic and highly integrated process. Hence, ancillary effects of viral infection could confound interpretation of a specific silencing event [2]. The nuclear-encoded 22-kD psbS protein is essential for regulated dissipation of excess quanta in PSII referred to as nonphotochemical quenching (NPQ) [8]. Mutation of psbS in Arabidopsis thaliana does not impair growth nor dramatically alter Photosystem I/II (PSI/PSII) processes other than NPQ [8-12]. We sought to determine if a comparable psbS deletion phenocopy could be created in Nicotiana benthamiana by VIGS and assess possible adverse effects of the systemic infection. Hence, untreated plants were compared to plants infused with the tobacco rattle virus silencing system (TRV-VIGS) containing either of two silencing targets: the N. benthamiana psbS gene or an extraneous bacterial gene sequence as a control.

A practical extension of VIGS-psbS is reintroduction of psbS as a basis for structure/function and PSII assembly studies. We demonstrate Agrobacterium-mediated transient expression of high levels of native N. benthamiana psbS that partially restores NPQ capacity. The latter results are discussed in terms of developmental aspects of photosynthetic gene expression as well as protein complex formation and turnover in relation to grana stacking.

2. Materials and Methods

2.1. Plant Growth Conditions

Nicotiana benthamiana was grown in 4-L pots on a nutrient-supplemented peat substrate in a growth chamber. The irradiance was 250 mmol quanta m⁻²·s⁻¹ with a light/dark cycle of 14/10 h, a temperature regime of 27˚C/20˚C, and relative humidity of 65%/90%.

2.2. Molecular Materials and Procedures

DNA constructs were prepared using standard procedures. Full-length N. benthamiana psbS cDNA (Gb EU645483) was generated by PCR using oligonucleotides NTABS1 and NTABS2 (Table 1), Qiagen Taq (Qiagen, Valencia, CA, USA), and DNA prepared from leaf RNA using M-MuLV reverse transcriptase (Roche, Indianapolis, IN, USA). The resulting DNA fragment was cloned into pCR2.1 (Invitrogen, Carlsbad, CA, USA) to generate plasmid pRH231. The tobacco rattle virus (TRV) system consists of two plasmids (pTRV1 and pTRV2-GATEWAY) with the engineered viral genome embedded in an Agrobacterium binary vector for efficient delivery in planta [13-15]. Target DNA for silencing RNA production was incorporated in the viral vector using the GATEWAY cloning system (Invitrogen). Five strains of Agrobacterium GV2260 were used containing the following plasmids: pTRV1 facilitates replication of the viral genome; pTRV2 with a chloramphenicol resistance marker (Cm^R) sequences; pTRV2 with the N. benthamiana phytoene desaturase gene (PDS); pDEST5 contained the pTRV2-GATEWAY vector with fulllength psbS cDNA from N. benthamiana; and pAV4 composed of pTRV2-GATEWAY containing the 184- base pair (bp) N. benthamiana psbS transit sequences [16]. Liquid cultures of pTRV2-containing Agrobacterium strains were each mixed 1:1 with strain YY192 containing pTRV1. Suspensions were injected into two fully expanded lower leaves of three-week-old N. benthamiana seedlings using a needleless hypodermic syringe. Measurements on newly emerged yet expanded leaves commenced two weeks after infusion.

Plasmid pRH231 was amplified with primers GATESPROF and GATESPROR or GATESPROF and GATESPROR2 and Qiagen Taq. The resulting DNA products were cloned into pDONOR207 as recommended with the GATEWAY cloning system (Invitrogen)

Table 1. Oligonucleotide

generating plasmids pENT5 and pAV3, respectively. These plasmids and pTRV2-GATEWAY were likewise treated as recommended to generate pDEST5 and pAV4, respectively.

Oligonucleotides NBTW1 and NBTW2 and separately NBTW3 and NBTW4 were amplified by PCR using the Expand High Fidelity system (Roche). The resulting products were joined using the same polymerase generating a 215-bp DNA fragment. Plasmid pRH231 was likewise amplified with oligonucleotides NBTW5 and NBEN20. This product was joined to the 215-bp DNA fragment to generate an 840-bp DNA fragment. The 840-bp fragment was cloned into pCR 2.1 (Invitrogen) generating plasmid pAV18. Plasmids pAV18 and pRH118 were cut with restriction endonuclease enzymes KpnI and BamHI and ligated. The resulting clone pNS451 includes the full-length N. benthamiana psbS cDNA under a CaMV35S promoter and nos termination sequences in the binary vector pCAMBIA1300 (CAMBIA, Canberra, Australia). DNA construct integrity was confirmed by sequence analysis.

Membrane protein fractions were prepared and probed for psbS by Western analysis as described previously [11]. The psbS content was related to a known mass of purified spinach psbS by densitometry. Levels of Lhca and Lhcb proteins were assessed immunologically (Agrisera). Chlorophyll (Chl) levels were measured in acetone extracts of leaf samples [17]. The density of Rubisco active sites was calculated after electrophoresis and densitometry of leaf extracts [18].

2.3. Gas Exchange and Optical Methods

The two-channel fast-response leaf gas exchange measurement system (Fast-Est, Tartu, Estonia) has been de scribed [17]. A portion of the attached test leaf was enclosed in the flow-through sandwich-type chamber (32 mm diameter, 3 mm height) and flushed with gas at a rate of 0.5 mmol·s⁻¹. The upper epidermis of the leaf was pasted with starch to a window separating the chamber volume from a thermostatting water jacket. The water jacket was maintained at 22˚C and heat budget calculations indicated that leaf temperature never exceeded 23˚C. Uptake of CO₂ was monitored with an infrared gas analyzer LI 6251 (LiCor, Lincoln, NE, USA) and a micro-psychrometer detected transpiration. Rates of CO₂ and H₂O exchange enabled calculation of dissolved CO₂ concentration at the carboxylation site considering stomatal and mesophyll diffusion resistances. Linear electron transport rate (J_C) associated with photosynthetic carbon metabolism was calculated as:

(1)

where A is the rate of net CO₂ assimilation (mmol·m⁻²·s⁻¹), R_K is Krebs cycle respiration in the light, K_s is the Rubisco CO₂/O₂ specificity factor, and C_c and O_c are the dissolved CO₂ and O₂ concentrations (mM) at the carboxylation sites [19]. The gas phase O₂ content was monitored using a calcia zirconia electrode Ametek S-3A (Thermox, Pittsburgh, PA, USA). Integration of the O₂ pulse following a saturating single turnover Xe flash at low background O₂ levels (10 to 50 mmol·mol⁻¹) provided a measure of PSII reaction center (RC) density [20].

All light beams were directed to the leaf by a fiber optic guide (Fast-Est, Tartu, Estonia). Actinic white light (WL) and fluorescence saturation pulses (10,000 mmol·quanta·m⁻²·s⁻¹ for 1.5 s) were provided by Schott KL 1500 sources. Measuring beams for fluorescence and 810-nm transmittance illuminated separate spots on the upper leaf surface. Far red light (FR, 50.6 mmol·quanta·m⁻²·s⁻¹, 720 nm) was provided by a feedback-stabilized light-emitting diode source (Fast-Est, Tartu, Estonia).

Chl fluorescence yield was measured with a PAM-101 equipped with an ED-101 emitter-detector unit (H. Walz, Effeltrich, Germany). Corrections for leakage of the measuring beam to the detector, detector oversaturation, fluorescence undersaturation during pulses, and PSI fluorescence were applied [21]. The quantum yield of PSII electron transport based on fluorescence (Y_F) and corresponding electron transport rate (J_F) are given by:

(2)

and

(3)

where F_s is the steady state and F_m is the pulse-saturated fluorescence yield and PAD (mmol·quanta·m⁻²·s⁻¹) is the photon absorption density [22]. The PSII partitioning coefficient (a_II) is estimated based on the quantum yields of electron flow to CO₂ (Y_C) and PSII electron transport (Y_F) in limiting light (LL) such that:

(4)

where

(5)

The corresponding partitioning coefficient for PSI (a_I) was likewise based on the optically-measured rate of electron flow from plastoquinol to PSI (J_I) and reduction level of P700 (P700_red) as:

(6)

where

(7)

Light absorption by non-photosynthetic chromophores (a_np) was calculated as 1 - a_I - a_II. Partitioning of FR quanta to PSI [a_I(FR)] was based on O₂ evolution (see above) assuming a PSII quantum efficiency of 0.8. NPQ is expressed in terms of the rate constant for regulated quenching of PSII excitation [23]:

(8)

Leaf transmittance at 810 nm was monitored using a single-beam photometer FS810-A (Fast-Est, Tartu, Estonia). Signal deconvolution has been described [24].

2.4. Experimental Protocols

Test plants were pre-darkened for 12 h to ensure full relaxation of NPQ. After mounting and equilibrating the test leaf in the chamber, dark respiration rate and the dark-adapted minimal (F_od) and maximal (F_md) fluorescence yields were recorded. Next, saturating single turnover flashes were applied to assess PSII RC density. The FR light titration procedure that followed enabled assessment of PSI donors and equilibrium constants [24]. Steady state levels of A, F_s, F_m, and the light-dark 810-nm absorption transient were recorded at incident irradiances of 2000, 1400, 760, 460, 260, 140, 75, 35, 13, and 0 mmol·quanta·m⁻²·s⁻¹ (360 mmol CO₂ mol⁻¹, 21% O₂). These quantities were then recorded at an irradiance of 2000 mmol·quanta·m⁻²·s⁻¹ and a gas phase of 2000 mmol CO₂ mol⁻¹ (HCHL conditions). Measurements were recorded at an irradiance of 760 mmol·quanta·m⁻²·s⁻¹ and CO₂ levels of 360, 80, 40, 0, 200, and 520 mmol·mol⁻¹. The O₂ concentration was reduced to 2% and measurements were recorded at CO₂ levels of 200, 100, 50, 0, and 200 mmol·mol⁻¹. An integrating sphere and microspectrometer (Ocean Optics, Dunedin, FL, USA) apparatus was used to assess the spectral transmittance of a 2-cm² disc from the chamber-enclosed area of the test leaf. Leaf absorption coefficients for WL and FR (a_WL and a_FR, respectively) were calculated considering the spectral emission profiles for these sources. The enclosed area of the test leaf (7.8 cm²) was excised, frozen in liquid N₂, and stored at −80˚C pending membrane protein extraction and pigment analysis.

3. Results

3.1. Virus Induced Gene Silencing of psbS Expression

The primary effect of VIGS is reduction in mRNA transcript level for the targeted gene. Reverse Transcriptase (RT) PCR established that psbS transcript levels were specifically lowered in N. benthamiana plants treated with TRV-VIGS-psbS (pDEST5, full-length psbS cDNA) compared to uninfused controls (Figure 1). This reduc-

(a)(b)

Figure 1. Assessment of psbS transcript levels. A. RT-PCR was performed using total RNA samples from leaves receiving no Agro-infiltration (uninfused 1-3) and from leaves inoculated with pTRV1 and pTRV2-NbpsbS (silenced 1-3). Samples from PCR cycles 18, 21, 24, and 27 amplified a 798-bp psbS-specific band and a 1.6-kb ubiquitin band (ubi) from cycles 27, 30, 33, and 36. B. An anti-psbS Western blot of leaf total membrane protein fractions compares psbS expression from uninfused N. benthamiana and from plants treated with a pTRV2 control construct (VIGS-Cm^R, see text) or pTRV2-NbpsbS (VIGS-psbS). We note that the response to VIGS-psbS was similar to the effect of deletion of psbS in Arabidopsis thaliana (A.t.).

tion in transcript level was associated with a loss of immunodetectable psbS (Figure 1, Table 2). As a positive control, we routinely confirmed the presence of VIGS in separate plants by targeting the phytoene desaturase gene (PDS), which causes bleaching of emerging leaves.

Application of VIGS typically results in a chimeric expression pattern for the targeted gene in plants [2]. We confirmed this with respect to psbS accumulation and expression of NPQ (Equation (8)). Figure 2 shows NPQ (panels A and B) and psbS levels (panels C and D) in leaves sampled 10 to 15 days after infusion with VIGSpsbS. Leaves already substantially developed at day zero exhibited the lowest level of additional expansion.

Younger leaves expanded relatively more (intermediate level) while leaves incipient at the time of inoculation were exposed to VIGS-psbS throughout their growth (100% expansion). Neither NPQ nor psbS levels in control (uninfused) leaves exhibited a clear effect of either sampling position along the long axis or leaf developmental stage for the low and intermediate expansion levels. However, NPQ was significantly elevated in the youngest leaves. In contrast, VIGS-psbS plants showed very low

Table 2. Summary of leaf photosynthetic parameters for normal and TRV-VIGS-Treated N. benthamiana.

NPQ in newly developed leaves and in the basal and middle regions of leaves that underwent intermediate expansion. The same pattern was evident in the distribution of psbS. Cell division and chloroplast maturation is most active in the basal region of the developing dicotyledonous leaf. Silencing of psbS expression is most evident in leaf tissue formed post-inoculation.

Table 2 confirms that co-suppression of psbS and NPQ was a specific result of TRV-VIGS treatment with psbS as the silencing target. Separate control plants (VIGS-Cm^R) were likewise treated with vector system containing a bacterial chloramphenicol resistance gene (kindly provided by S. P. Dinesh-Kumar) in the target domain to test for effects of proliferation of a non-silencing TRV-VIGS

Conflicts of Interest

The authors declare no conflicts of interest.

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