Role of unfolded protein response in plant virus infection

Abstract

A new study of Potato virus X (PVX) revealed that a viral movement protein, named TGBp3, triggers the unfolded protein response (UPR) which monitors accumulation of aberrant proteins the endoplasmic reticulum (ER) and targets them for degradation. The PVX TGBp3 resides in ER and activates bZIP60, a transcription factor involved in the UPR pathway. Knockdown of bZIP60 hampers virus infection in protoplasts and whole plants. Preliminary evidence indicates that UPR regulates cellular cytotoxicity that could otherwise lead to cell death if the PVX TGBp3 reaches high levels in the ER. SKP1 expression appears to be linked to bZIP60 and is a component of the SCF-complex mediating proteasomal degradation of cellular substrates. SKP1 expression is induced by PVX TGBp3 and plays a role in regulating PVX spread in whole plants. We propose that SKP1 might be linked to TGBp1-mediated degradation of AGO1.

Various cellular disturbances cause build-up of unfolded proteins in the ER, prompting a response that is conserved across kingdoms, known as the unfolded protein response (UPR). Proteins that fail to fold correctly or become inappropriately glycosylated are not deployed to distal compartments or to the secretory system, but are rapidly degraded. UPR functions during normal processes as well as in stressful situations.¹ Environmental stress such as salt or pathogen attack can trigger vigorous protein synthesis or protein transfer to the Golgi.^2,3 This leads to the increase of misfolded proteins in the ER and triggers the UPR. Thus the purpose of the UPR is to restore normal ER function and prevent the potential cytotoxic impact of malformed proteins.⁴ Elimination of malformed proteins in the ER is achieved by export to the cytosol for degradation via the ubiquitin-proteasome pathway (Fig. 1).

Figure 1.

In plants IRE 1 and several bZIP factors are identified as transmembrane sensors of ER stress. BiP binds transmembrane sensors under normal conditions. In response to accumulation of aberrant proteins in the ER BiP releases its hold on the sensors which activates several transduction pathways. bZIP60 is proteolytically processed and the truncated version is translocated to the nucleus where it activates expression of UPR related genes including ER resident sensors. The branch of the pathways relating to proteasome activation and targetted protein degradation has not yet been elucidated in plants.

A variety of ER resident sensors are activated by ER stress.⁵ IRE1 is one such ER-resident sensor that upon activation splices the bZIP transcription factor XBP1 (HAC1) mRNA in mammalian cells.^6,7 XBP1 (HAC1) is translated and subsequently activates the transcription of a group of genes that possess UPR cis-activating regulatory elements (ERSE) in their promoter regions. In Arabidopsis the IRE1 homologues identified plant specific (p-UPRE) cis elements in promoter regions.⁸ This IRE-1 pathway is the first example of a protein signal transduced from the ER to the nucleus. Additional ER resident sensors/transducers have been identified, some of which are conserved, and others that are unique to each organism.

The UPR mechanism (Fig. 1) involves increasing synthesis of certain ER-resident proteins needed to restore proper protein folding such as the ER luminal binding protein (BiP), protein disulfide isomerase (PDI), calreticulin (CRT) and calmodulin (CAM).^5,9 Overexpression of certain ER resident chaperones has been shown to be effective for restoring plant vigor.² For example, plants that overexpress BiP from soybean exhibit improved drought response.¹⁰ The UPR pathway has been found to play a role in salt and osmotic tolerance, which has a major impact on plant growth and crop production.

Viral-Induced Modifications of Membranous Networks are Well-Documented

Viral protein interactions with the ER are often vital contributions to persistent infection. For many positive strand RNA viruses, the ER provides architectural support for genome synthesis and translation.¹¹ Viruses such as Potato virus X (PVX) Cowpea mosaic virus (CPMV), Brome mosaic virus, Red clover necrotic mosaic virus, Peanut clump virus (PCV) stimulate de novo membrane synthesis which is essential for virus replication and results in morphological changes in the cortical ER network.^12–16 Cerulenin is an inhibitor of de novo lipid synthesis. Protoplasts treated with cerulenin show reduced levels of CPMV, PVX and PCV replication indicating that formation of new membranes plays an essential role in the replication of these viruses.^16–18

Real time RT-PCR analyses revealed an increase in ER resident chaperones which we initially hypothesized could due to expansion of the ER network or that increased levels of chaperones are needed in response to PVX infection.^15,16,19 Thus, if virus infection increases membrane synthesis it is reasonable to suspect that the molecular markers within the ER network would also increase. To determine if expansion of the ER network was sufficient to explain the increases in gene expression relevant to accumulating ER-resident proteins, we conducted further real time RT-PCR analysis to determine if there was a global increase in gene expression relating to the secretory pathway. In particular we examined BiP, SYP21, SYP41 and SYP61 as molecular markers which are located in the ER, trans-Golgi network and prevacuolar compartments (Fig. 2). We compared RNA accumulation in healthy leaves and PVX-GFP infected N. benthamiana leaves at 3, 6 and 9 days post inoculation (dpi). PVX infection leads to a general increase in population values (representing fold-changes in gene expression) for BiP at 9 dpi (Fig. 2). The median values at 9 dpi are 10- to 12-fold higher than healthy samples. However there was no change in the median or population values for SYP21, SYP41 or SYP61. Notably, these data are collected from three samples. In Ye et al. (2011) BiP expression was analyzed using a population of 20 infected plants and non-parametric analysis was conducted to ascertain the median value among these plants. The analysis reported in Figure 2 is an average of three plant samples. The prior analysis was of a large population, and while the median values were lower, the values obtained in this smaller experiment, fall within the range of values obtained from the larger population.¹⁹ These data argue that PVX infection did not require synthesis of molecular markers occurring throughout the ER and secretory pathway. Thus, if membrane synthesis and expansion is occurring during PVX infection, we cannot state that there is a concomitant change in ER-Golgi network markers.

Figure 2.

Gene induction following inocualtion of PVX to N. benthamiana leaves. Bars represent average of three samples collected at 3, 6 and 9 d post inocualtion. Healthy untreated sample is included as a control. Mock inoculated (treated with agro-infiltration buffer) was used as a control and calibrator sample. Total RNA was isolated from samples and first strand cDN A synthesis was carried out as described in Ye et al. (2011).¹⁹ qPCR was carried out using 25 µl reactions and 100 to 900 nM primers designed using the coding sequences for known N. benthamiana genes. Initial RT-PCR tests of the gene specific primers confirmed their ability to amplify single bands of the predicted sizes from N. benthamiana cDNA. With the Power SYBR Green II Master Mix and ABI 7500 PCR machine (Applied Biosystem, Foster City, CA), 25 ng cDNA was used to perform qPCR. Reactions were incubated first at 95°C for 10 min, and then 40 cycles of 95°C for 15 sec and 60°C for 60 sec. The comparative C_T method was employed for relative quantitation of gene expression following virus treatments. qRT-PCR efficiencies were determined by control amplifications using 0.01, 0.1, 1, 10, 100 ng of template cDNA. Duplicate PCR reactions for each sample were carried out and averaged. The comparative C_T method employs the formula 2^{−dd CT} where the values of the endogenous control (18S RNA) and calibrator (constant quantity of healthy sample template) and are subtracted from the target sample value to provide the ddC_T value. The 2^{−dd CT} represents the fold of RNA accumulation.

Based on these observations, the significant increase in BiP, led us to believe that this chaperone is providing an activity necessary for virus infection. In addition, given the number of PVX genes that are known to associate with the ER (replicase, TGBp2 and TGBp3) it is possible that one of these genes triggers UPR and BiP gene expression. Later we identified bZIP60 as a transcription factor that upregulates UPR BiP expression in response to the PVX TGBp3 elicitor.

A basic-region Leucine Zipper (bZIP) Transcription Factors which is Essential for UPR is Linked to PVX Infection

Researchers exploring UPR induction using chemical or salt treatment have reported that bZIP gene expression is induced about 3-fold.^3,7 Three bZIP transcription factors possessing transmembrane domains have been linked to UPR regulation: AtbZIP17, AtbZIP28 and AtbZIP60.²⁰ ER stress related transcription factors recognize promoters containing P-UPRE or ERSE cis-elements. Following nuclear translocation, transcription factors serve to upregulate expression of certain ER resident chaperones, such as BiP and PDI, which are involved in maintaining ER homeostasis in response to stress inducing events.^8,20–22 At the same time, the misfolded proteins in the ER are exported to the cytosol where plant ubiquitin ligase complexes recruit target proteins for degradation by the 26S proteasome. Importantly, the link between plant bZIP transcriptional activation and ubiquitin-proteasome pathway has not been elucidated (see Fig. 1).

In an exciting new report in Plant Physiology (Ye et al. 2011) we report evidence that the membrane bound PVX TGBp3 regulates UPR in a manner that promotes persistent virus infection. The data reveal that PVX or TGBp3 by itself upregulates bZIP60, certain ER resident chaperones, and SKP1 which is a component of SCF ubiquitin ligase complexes. We examined the effects of silencing bZIP60 in protoplasts and plants. Gene expression was inhibited to a greater extent in protoplasts that were silenced by delivery of double stranded RNAs (dsRNAs) than in plants that were silenced using the Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) to knock down expression of bZIP60. Thus, when bZIP60 expression was greatly inhibited in protoplasts, we noted that PVX accumulation was blocked, suggesting that bZIP60 is a factor in virus replication. These data argue that TGBp3, although a movement protein, contributes to the regulation of virus replication by its impact on host gene expression. In whole plants, where bZIP60 expression was knocked down by 77% below mock treated plants, we noted that the spread of PVX infection was hampered. If we compare the silencing methods employed in protoplasts and plants, the TRV VIGS system does not adequately eliminate bZIP60 expression that would produce results comparable with the dsRNA delivery to protoplasts. Considering both data, minimal levels of bZIP60 are needed to initiate infection in plants. A more effective method to eliminate bZIP60 expression in plants could be useful in developing a resistance strategy to PVX infection.

UPR is reported to be an early response to pathogen invasion in anticipation of the increase in protein synthesis along the ER.² In particular, BiP is a well known component of cellular cytoprotective responses to alterations in the ER or accumulation of misfolded proteins and controls the status of certain UPR transmembrane signal transducers (e.g., IRE1, PERK and ATF6).^1,6 BIP is known to be upregulated by bZIP60 and is also reported to regulate cytotoxic damage to cells.²³ Therefore, we delivered each viral gene by agro-infiltration to plant cells to learn if the accumulation of any one PVX protein could lead to cytotoxic cell death. PVX TGBp3 appeared to induce a hypersensitive response that was alleviated by the co-delivery of NbBLP-4 (BiP homologue) via agro-infiltration. Thus, we can conclude one role for UPR is to reduce cytotoxicity that may lead to necrosis as viral proteins accumulate inside cells.

SKP1 is a Component of SCF Family of E3 Ubiquitin Ligases and a Factor in PVX Infection

We also reported that silencing SKP1 reduced the number of infection sites on inoculated leaves and the number of infected plants suggesting that it plays a role in PVX infection. Although we failed to ascertain its exact role in PVX pathogenesis, SKP1 is an essential component of the SCF family of E3 ubiquitin ligases which provides substrate ubiquitination preceding proteasome-mediated degradation.^24,25 Typical substrates are cellular proteins crucial for eukaryotic physiology and defense. Recent work on the polerovirus Beet western yellows virus (BWYV) showed that the viral P0 protein binds SKP1 and targets AGO1, which is the effector nuclease of RNA silencing machinery, for degradation via a mechanism that involves the 26S proteasome.^26,27 Silencing SKP1 confers BWYV resistance indicating that this gene is also a factor in polerovirus infection.²⁷

An intriguing possibility is that TGBp3 upregulates SKP1 to enable proteasomal degradation of AGO1. The PVX TGBp1 silencing suppressor targets AGO1 for degradation via the proteasome.²⁸ Thus combining this work with previous reports of TGBp1, it is possible that TGBp3 upregulates UPR and components of the proteasome pathway to enable degradation of AGO1 mediated by TGBp1. Thus it is worth considering that the TGBp1 and TGBp3 act in concert to regulate host defense and stress responses in a manner that renders plants more susceptible to PVX infection. Further research is needed to determine the link between TGBp1, TGBp3 and proteasomal degradation of the silencing machinery.

Addendum to: Ye C, Dickman MB, Whitham SA, Payton M, Verchot J. The unfolded protein response is triggered by a plant viral movement protein. Plant Physiol. 2011;156:741–755. doi: 10.1104/pp.111.17411.