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Photosynthesis‐related genes induce resistance against soybean mosaic virus: Evidence for involvement of the RNA silencing pathway

Abstract Increasing lines of evidence indicate that chloroplast‐related genes are involved in plant–virus interactions. However, the involvement of photosynthesis‐related genes in plant immunity is largely unexplored. Analysis of RNA‐Seq data from the soybean cultivar L29, which carries the Rsv3 resistance gene, showed that several chloroplast‐related genes were strongly induced in response to infection with an avirulent strain of soybean mosaic virus (SMV), G5H, but were weakly induced in response to a virulent strain, G7H. For further analysis, we selected the PSaC gene from the photosystem I and the ATP‐synthase α‐subunit (ATPsyn‐α) gene whose encoded protein is part of the ATP‐synthase complex. Overexpression of either gene within the G7H genome reduced virus levels in the susceptible cultivar Lee74 (rsv3‐null). This result was confirmed by transiently expressing both genes in Nicotiana benthamiana followed by G7H infection. Both proteins localized in the chloroplast envelope as well as in the nucleus and cytoplasm. Because the chloroplast is the initial biosynthesis site of defence‐related hormones, we determined whether hormone‐related genes are involved in the ATPsyn‐α‐ and PSaC‐mediated defence. Interestingly, genes involved in the biosynthesis of several hormones were up‐regulated in plants infected with SMV‐G7H expressing ATPsyn‐α. However, only jasmonic and salicylic acid biosynthesis genes were up‐regulated following infection with the SMV‐G7H expressing PSaC. Both chimeras induced the expression of several antiviral RNA silencing genes, which indicate that such resistance may be partially achieved through the RNA silencing pathway. These findings highlight the role of photosynthesis‐related genes in regulating resistance to viruses.


Symptoms of viral infection in plants usually include a change in
the green pigmentation such as mottling, mosaic, chlorosis, and yellowing. Most of these symptoms indicate changes in photosynthetic activity in the infected plants (Liu et al., 2020;Scholthof et al., 2011). It has long been known that viral infection leads to reduced photosynthesis and major changes in chloroplast ultrastructure (Bhattacharyya & Chakraborty, 2018;Lehto et al., 2003). The roles of chloroplasts in virus replication, virus movement, and plant defence have only recently been investigated (Azim & Burch-Smith, 2020;Bhattacharyya & Chakraborty, 2018;Ganusova et al., 2020;Zhao et al., 2016).
Photosynthesis includes two major stages: a light-dependent stage and a light-independent stage. In the light-dependent stage, photosystem I (PSI), cytochrome, photosystem II (PSII), and ATPase synthesis sequentially contribute to the production of NADPH and then ATP, which are used in the light-independent stage to produce sugar through the Calvin cycle (Moejes et al., 2017;Nevo et al., 2012;Yu et al., 2020). Virus interference with chloroplasts in general, and with photosynthesis in particular, can occur on different levels.
Because the chloroplast is the site for the biosynthesis of several defence-related hormones and helps control plasmodesmata (PD) permeability, some viruses reduce host defences by targeting the chloroplast with specific viral proteins (Alazem & Lin, 2015Ganusova et al., 2020). The P25 protein of potato virus X (PVX), for example, interferes with the function of ferredoxin 1 (FD1), an important protein involved in electron transfer between PSII and PSI, resulting in reduced levels of the defence-related hormones abscisic acid (ABA) and salicylic acid (SA) . This reduction decreases callose accumulation at PD, and consequently increases PD permeability and PVX spread in the host plant . Because the chloroplast is also the site for the replication of several RNA viruses, viral effectors are expected to recruit specific chloroplast proteins into their viral replication complex (Budziszewska & Obrepalska-Steplowska, 2018;Cheng et al., 2013;Ganusova et al., 2020;Zhang et al., 2017). Bamboo mosaic virus (BaMV), for example, recruits the chloroplast phosphoglycerate kinase (chl-PGK) protein, that is, the viral RNA genome binds to chl-PGK and transports it to the chloroplast (Cheng et al., 2013). Once in the chloroplast, BaMV recruits further chloroplast proteins into the viral replication complex to complete the infection cycle (Huang et al., 2017). In another example, infection with rice stripe virus (RSV) dramatically changes the proteome profiles of the Nicotiana benthamiana protoplast and chloroplast, resulting in a significant decrease in the number of nuclear-encoded chloroplast-localized proteins; the decrease is caused by RSV interference with three host factors (K4CSN4, K4CR23, and K4BXN9) that are involved in protein delivery to the chloroplast (Zhao et al., 2019).
It follows that viral interference with the functions of chloroplast proteins explains why photosynthesis is reduced in susceptible plants (i.e., in compatible interactions). In contrast, some resistant plants show increased expression of photosynthesis-related genes.
For example, expression of photosynthesis-related genes in soybean cultivar L29 (which carries the resistance [R]-gene Rsv3) was increased in response to infection by the avirulent G5H strain but not in response to the virulent G7H strain of soybean mosaic (SMV) (Alazem et al., 2018).

Soybean mosaic virus is a member of the genus Potyvirus and
has a single-stranded, positive-sense RNA genome that encodes 11 viral proteins and is about 10 kb in length (Hajimorad et al., 2018;Liu et al., 2016). SMV has many strains distributed worldwide and, depending on the phenotypic responses of various soybean cultivars, these strains have been classified into seven distinct strains in the United States (G1 to G7) and into 21 strains in China (SC1 to SC21) (Hajimorad et al., 2018). Genetic resistance to SMV is mainly achieved through different strain-specific NLR-type R-genes such as the Rsv and the Rsc groups (Widyasari et al., 2020). There are several other non-NLR host factors that have been found to be critical for resistance, either because they are key components in the signalling pathway downstream of the R-gene or because they are part of a plant system that degrades viral RNA or protein (i.e., antiviral RNA silencing and double-stranded RNA ribonuclease) (Ishibashi et al., 2019;Liu et al., 2014;Widyasari et al., 2020).
Here, we investigated the roles of two photosynthesis-related proteins, PSaC and ATPsynα, in the resistance to SMV in soybean cultivar L29, which is resistant to G5H but not to G7H. Both proteins were strongly up-regulated in cultivar L29 in response to G5H, whereas the response to G7H infection was rather weak. However, their roles in resistance to SMV have not been investigated. PSaC or ATPsynα, which may account for the resistance phenotype induced by both genes.

| Chloroplast-relatedgenesareinducedin the resistant cultivar L29 in response to SMV-G5H infection
The soybean cultivar L29 carries the R-gene Rsv3, which confers resistance against the SMV avirulent strain G5H but is ineffective against the virulent strain G7H ). We previously obtained RNA-Seq data from L29 plants infected with strains G5H and G7H (Alazem et al., 2018). The data showed that, in the incompatible interaction (resistance against G5H), a large number of differentially regulated genes were photosynthesisrelated (Alazem et al., 2018). To examine this list more closely, we searched for the top up-regulated genes (fold change >1) that were induced only in response to G5H infection at any time point (Figure 1a). Most of these genes have different functions related to photosynthesis/chloroplasts (Table 1). While the expression of most of these genes was induced in response to G5H, the expression of several was temporarily and slightly increased in response to G7H at 8 h postinfection (hpi) but then decreased at 24 and 54 hpi (Figure 1a). This suggests a possible relationship between their suppression and G7H virulence. We selected two F I G U R E 1 Expression of photosynthesis-related genes in response to soybean mosaic virus (SMV) infection. (a) Heat-map of photosynthesis-related genes regulated by infection with the avirulent strain G5H or the virulent strain G7H of SMV. Expression of ATPsynα (b) and PSaC (c) in L29 plants (which carry the Rsv3 resistance gene) at 8, 24, and 54 h postinfection (hpi) by G7H::eGFP. Expression of ATPsynα (d) and PSaC (e) in Lee74 plants (rsv-null) at 8, 24, and 54 hpi by G7H::eGFP. Actin11 was used as the internal control. In (b-e), values are means + SD of three biological replicates. Values were compared to that of the corresponding mock-treated plants (the bar on the left) with one-sided Student's t tests; * and ** indicate a significant difference at p < 0.05 and p < 0.01, respectively TA B L E 1 Functional analysis and gene ontology of the photosynthesis-related genes regulated by SMV infection genes, Glyma.18G155300.1 and Glyma.12G232000.1, which were strongly down-regulated in response to G7H but up-regulated in response to G5H (Figure 1a), for further analysis. In the soybean DB (Soybase) assembly 4 v. 1, Glyma.18G155300.1 and Glyma.12G232000.1 were reported to encode the PSaC subunit of the PSI subunit (PSaC) and the ATP-synthase α-subunit (ATPsynα), respectively (Table 1) (Brown et al., 2021;Grant et al., 2010).
To confirm the RNA-Seq data, we used reverse transcription quantitative PCR (RT-qPCR) to measure the expression of both genes in L29 plants infected with G7H. Expression of GmATPsynα significantly increased at 8 hpi but then declined at 24 and 54 hpi to levels comparable to that in mock treatments ( Figure 1b). GmPSaC Glyma.12G232000.1 will be referred to as GmPSaC and GmATPsynα, respectively.

| GmPSaC and GmATPsyn-α genes are induced in cultivars with different resistance backgrounds
The finding that GmPSaC and GmATPsynα are temporarily induced in L29, which is immune to G5H via the Rsv3 gene but is susceptible to G7H, prompted us to determine the expression of both genes in other cultivars with different resistance backgrounds. For this, three rsv-null cultivars (Lee74, W82, and SMK), one Rsv4 cultivar (V94), and one Rsv3 cultivar (L29) were assessed for their susceptibility to G7H.
Infection by G7H (which expresses green fluorescent protein, GFP) induced visual symptoms in the systemically infected leaves (SL) of all cultivars except V94 at 10 days postinoculation (dpi) (Figure 3a).
Confirming this, a protein blot revealed that GFP from G7H was un-

| OverexpressionofATPsyn-αandPSaC inducesresistanceagainstG7Hinthesusceptible cultivarLee74
To determine the effect of ATPsynα and PSaC on resistance to G7H, the coding sequence (CDS) of each gene was cloned from L29 plants into the G7H genome to create pSMV-G7H::eGFP::ATPsynα and pSMV-G7H::eGFP::PSaC constructs ( Figure 4a). As a member of the Potyvirus genus, SMV uses the host's cellular translation machinery to translate its RNA into one single polyprotein, which undergoes self-cleavage to produce 11 different viral proteins (Hajimorad et al., 2018). We previously took advantage of this characteristic by inserting Green fluorescent protein (GFP) as a reporter gene within the SMV-infectious clone pSMV-G7H::eGFP (Seo et al., 2014). Here, we inserted both genes downstream of the GFP within the G7H genome ( Figure 4a). The rsv-null cultivar Lee74 was rubinoculated at the unifoliate stage with plasmids of both constructs (the seedlings were about 12 days old) and the accumulation level was measured in IL and SL at 7 and 14 dpi, respectively. While western protein blot showed that both genes were translated into proteins and that they were not lost or missed in the translation of the SMV polyprotein in the SL ( Figure S3d). In addition, the expression of these genes in pSMV-G7H::eGFP might trigger their silencing in plants. To examine this, RT-qPCR with primers annealing to the 3′ untranslated regions of both genes showed that endogenous transcripts of both genes were not affected by the constitutive expression via pSMV-G7H::eGFP ( Figure S4a,b). To determine whether this resistance is connected to ROS, 3,3′-diaminobenzidine staining on the IL 7 dpi showed no ROS in response to G7H::eGFP or the constructs expressing either gene (Figure 4e). This indicated that ROS may not be part of the resistance induced by PSaC or ATPsynα.

| KnockdownofATPsyn-α and PSaC increased Lee74susceptibilityagainstG7Hinfection
To confirm the role of both genes in resistance against G7H, virus- to that of PSaC-silenced plants (Figure 5c). RT-qPCR and western blot for eGFP confirmed that G7H::eGFP accumulated more in the ATPsynα knocked-down plants, and that G7H accumulation level was similar between BPMV-EV and BPMV-PSaC plants (Figure 5d,e).
These data indicated that silencing ATPsynα has a strong influence on plant susceptibility to G7H infection, unlike that of PSaC, which was similar to the control BPMV-EV treatment.
To determine whether the silencing process may affect offtarget transcripts, a BLAST search using both genes was made in the Soybase database in a search for paralogs. Only ATPasesynα had two close paralogs: Glyma.16G115300.1 (which encodes a chloroplast ATP synthase subunit α) and Glyma.05G092300.1 (which encodes a mitochondrial ATP synthase subunit α). However, the designated fragment for silencing shares low similarity with the two paralogs ( Figure S5). Expression levels of either gene were not affected by the silencing of ATPsynα ( Figure S6a,b), which indicates that silencing probably did not affect off-target transcripts.

| LocalizationofATPsyn-αandPSaCin N. benthamiana and their effects on N. benthamiana resistanceagainstSMV-G7H
To investigate the localization of ATPsynα and PSaC, we expressed both genes in the binary vector pBin61-HA-mCherry . We used the chloroplast-localized protein from Arabidopsis, EMB1303, fused with eGFP as a marker protein (Huang et al., 2009).
AtEMB1303 localized in the chloroplast membrane, and the GFP signal was also detected in the extended stromules ( Figure 6a and

| Involvementofdefence-relatedhormonesin ATPsyn-α-andPSaC-mediatedresistance
The chloroplast plays a critical role in plant immunity because GmDREB1A-2 (h). The unifoliate leaves of Lee74 plants were inoculated with pSMV-G7H::eGFP expressing ATPsynα or PSaC genes (pSMV-G7H::eGFP::ATPsynα or pSMV-G7H::eGFP::PSaC, respectively); the inoculated leaves (IL) and systemically infected leaves (SL) were collected at 7 and 14 days postinoculation, respectively. Actin11 was used as the internal control. Values are means + SD of three biological replicates. Statistical analysis was carried out as described in Figure 1; * and ** indicate a significant difference at p < 0.05 and p < 0.01, respectively. An additional t test was carried out to compare expressions in the pSMV-G7H::eGFP::ATPsynα and pSMV-G7H::eGFP::PSaC treatments to that in pSMV-G7H::eGFP than in plants infected with pSMV-G7H::eGFP. Such an increase was only recorded for PAD4 in the SL of plants infected with both constructs (Figure 7a,b). Similarly, the expression levels of the JA-

| AntiviralRNAsilencinggenesareregulatedin ATPsyn-α-andPSaC-mediatedresistance
Because SA and ABA affect the expression of RNA silencing genes (Alazem & Lin, 2020)  TMV accumulation and pathogenicity were greatly enhanced, indicating that ATPsynγ is involved in limiting the intracellular trafficking of TMV as well as in inducing defence signalling pathways (Bhat et al., 2013). Interestingly, an opposite effect was found for ATP-synγ in response to infection with PVX or tomato bushy stunt virus, that is, their spread was decreased in ATP-synγ-silenced plants (Bhat et al., 2013). In another example, infection with potato virus Y reduced the photosynthesis rate through the HC-Pro protein in Nicotiana tabacum plants; HC-Pro interacted with the ATPsynβ subunit but did not affect the enzymatic activity of ATP synthase, leading to a reduced ATP synthase content in HC-Pro-transgenic plants (Tu et al., 2015).
In other words, we cannot generalize about the effects of ATPsyn subunits on host plant resistance to viruses; the influence on resistance can vary depending on the virus group.
ATPsynα andβ form the hydrophilic head (cF1) powered by the membrane-embedded-cF0 rotary motor in the ATP synthase complex. ATPsynα guides protons to and from the c-ring protonation site (Hahn et al., 2018). In general, ATP synthase is redox-regulated and controlled by the chloroplast thioredoxin system, which is connected with photosynthesis (Hisabori et al., 2013). Regulation of redox controls the accumulation of ROS and nitrogen species, both of which are important for resistance against several pathogens (Bentham et al., 2020;Frederickson Matika & Loake, 2014).
Given the absence of necrotic lesions in soybean expressing PSaC or ATPsynα, however, it is unlikely that ROS is involved in ATPsynαor PSaC-mediated-defence against SMV-G7H.
PSaC encodes a subunit in the PSI complex and functions in electron transfer and ferrodoxin docking on the stromal side of PSI (Rantala et al., 2020). Although studies on the role of PSaC in plantvirus interactions are lacking, a previous report indicated a positive role for another member of the PSI complex, PSaK, in resistance against plum pox virus (PPV) (Jimenez et al., 2006). Infection with PPV decreased PSaK expression in N. benthamiana, and when PSaK was knocked down, PPV accumulation was enhanced. In addition, the cylindrical inclusion protein of PPV interacted with PSaK and possibly interfered with its function (Jimenez et al., 2006). Our data showed that, in response to SMV-G7H infection, expression of PSaC and ATPsynα increased in resistant soybean plants but did not decrease in susceptible plants (Figure 3d,e). That their overexpression reduced SMV-G7H accumulation (Figure 4b,d) suggests that both genes partially contributed to resistance against SMV. In line with this finding, silencing ATPsynα, but not PSaC, increased soybean susceptibility to SMV-G7H infection ( Figure 5). This confirms the role of ATPsynα in resistance, but also suggests functional redundancy for genes might interrelate with PSaC, which could be members of the PSI.
The resistance conferred by ATPsynα is stronger than that conferred by PSaC in both N. benthamiana and soybean plants (Figure 4b,d). This could be attributed to the simultaneous induction of several genes in the defence signalling pathways of SA, JA, and ABA in response to pSMV-G7H::eGFP::ATPsynα, but for pSMV-G7H::eGFP::PSaC the response was limited to SA and JA ( Figure 7). Pathways of all of these hormones are involved in soybean resistance to SMV (Alazem et al., 2018(Alazem et al., , 2019Zhang et al., 2012). In fact, the connection between defence hormones and the antiviral RNA silencing pathway is well established (Alazem et al., 2019;Alazem & Lin, 2015. We previously showed that SA and ABA enhance the expression of the antiviral RNA silencing genes in soybean and A. thaliana, and that the enhanced expression confers partial resistance against SMV, BaMV, and PVX (Alazem et al., 2017(Alazem et al., , 2019. Our current findings show that ATPsynα induced the expression of more genes (DCL4a, RDR2a, and RDR6a) in the antiviral RNA silencing pathway than PSaC, which only induced the expression of RDR2a and only in the IL ( Figure 8). It is therefore likely that the stronger resistance triggered by ATPsynα than PSaC is due to the greater influence of ATPsynα on the antiviral RNA-silencing genes.
Because trafficking through PD is strongly regulated by light and the circadian clock (Brunkard & Zambryski, 2019;Ganusova et al., 2020), it is highly probable that chloroplast-related genes can adversely affect viruses in two ways, that is, the gene products may hinder cell-to-cell trafficking through PD and may also induce defence-related hormone signalling pathways. Our results provide evidence that induction of these photosynthesis genes induces hormone signalling pathways that eventually trigger antiviral RNA silencing pathways that partially contribute to local and systemic resistance to SMV (Figure 8). Whether SMV trafficking through PD is affected by photosynthesis genes requires further investigation. The effect of enhanced photosynthesis on plant resistance to viruses is incompletely understood and also warrants additional research.
We expected to detect ATPsynα and PSaC inside the chloroplast, but, surprisingly, we found that they were localized in the chloroplast envelope. In addition, both proteins were localized in the cytoplasm and the nucleus (Figure 6b,c). We did not detect any degradation of either protein by western blot (Figure 6d), which indicates that both proteins can be distributed to the cytoplasm and the nucleus for further functions that remain to be examined.
In conclusion, strong photosynthesis can increase resistance against viruses. Additional research is needed to clarify how chloroplasts in general, and photosynthesis in particular, enhance resistance against plant viruses.

ATPsyn-α and PSaC genes
The CDS of ATPsynα and PSaC genes were amplified and cloned from several soybean cultivars and were then cloned into a TA vector (pGEM-T Easy; Promega). The clones were confirmed by sequencing with gene-specific primers (Table S1). The CDS of both genes from L29 plants were then cloned into the pSMV-G7H::eGFP infectious clone to generate pSMV-G7H::eGFP::ATPsynα and pSMV-G7H::eGFP::PSaC as previously described (Seo, Lee, Choi, et al., 2009).
About 15 dpi, a pool of SL from three plants was mixed and divided into 0.1-g portions as a source of virus inoculum. After each 0.1-g portion was ground into powder in liquid nitrogen, it was mixed with 1 ml of phosphate buffer. The mixture was placed on ice for 10 min and was then centrifuged for 10 min at 4°C and 13,580 × g.

| SilencingATPsyn-α and PSaC in soybean plants
The BPMV silencing vector was used to silence ATPsynα and PSaC genes in Lee74 plants. In brief, fragments of 173 bp from PSaC CDS, and 347 bp from ATPsynα CDS were cloned in the antisense direction in the multiple cloning site of RNA2 of BPMV, as described previously (Zhang et al., 2010). Ten micrograms of BPMV plasmids (RNA1 and RNA2) were rub-inoculated onto the first unifoliate leaves of Lee74 plants, and the silencing efficiency was tested at 14 dpi in the second trifoliate leaf. The same leaf was sapinoculated with G7H::eGFP as described in section 4.2. Samples were collected from the SL 10 days after G7H::eGFP infection for further analyses.

| RNAextractionandRT-qPCR
Total RNA was extracted using TRIzol (Sigma) following the manufacturer's instructions. A 1μg quantity of total RNA was used for cDNA synthesis using the GoScript kit (Promega). RT-qPCR was carried out with SYBR Green (Promega) to measure the relative expression of target genes using the ΔΔC t method. Actin11 was used as an internal control, and the primers used in this study are listed in Table S1. One-sided Student's t tests (p < 0.05) were used to determine whether the expression level of each gene in each line was up-regulated or down-regulated relative to the mock-treated plants.

| Statisticalanalysis
RT-qPCR was carried out in three biological replicates, and each biological replicate was repeated in three technical replicates. In Figures   1 and 3

| Westernproteinblot
Total protein was extracted from 0.1 g of tissue collected from a pool of IL and SL from three plants, as described previously (Alazem et al., 2018). Constructs expressing GFP were detected by western blot using polyclonal anti-GFP antibody, and those expressing HA were detected using monoclonal anti-HA antibody (Sigma); Ponceau S staining was used on the loading control.
The phylogenetic trees were generated using MEGA 7.0 software and by applying the neighbour-joining method (Kumar et al., 2016).

| SubcellularlocalizationofATPsyn-αand PSaC proteins
ATPsynα and PSaC were cloned into the binary vector pBin61-3HA-mCherry . Agrobacterium infiltration was carried out on N. benthamiana plants using Agrobacterium tumefaciens C58C1 at OD 600 = 0.5, with the aid of 2b, the viral suppressor of RNA silencing (pPZP-2b), to enhance the expression of both genes.
Infection with pSMV-G7H::eGFP was carried out 1 day after agroinfiltration using 50 µl of infectious sap extract/leaf. Samples were collected at 3 dpi for confocal microscopy, and at 5 dpi for protein and RNA analysis. The chloroplast marker protein gene AtEMB1301 was cloned into pBin-eGFP and used as a marker for the localization of ATPsynα and PSaC proteins.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.