Identification and functional analyses of host factors interacting with the 17-kDa protein of Barley yellow dwarf virus-GAV

Barley yellow dwarf viruses (BYDVs) cause significant economic losses on barley, wheat, and oats worldwide. 17-kDa protein (17K) of BYDVs plays a key role in viral infection in plants, whereas the underlying regulation mechanism of 17K in virus infection remains elusive. In this study, we determined that 17K of BYDV-GAV, the most common species found in China in recent years, was involved in viral pathogenicity. To identify the host factors interacting with 17K, the full length coding sequence of 17K was cloned into pGBKT7 to generate the bait plasmid pGBKT7-17K. 114 positive clones were identified as possible host factors to interact with 17K through screening a tobacco cDNA library. Gene ontology enrichment analysis showed that they were classified into 35 functional groups, involving three main categories including biological processes (BP), cellular components (CC), and molecular functions (MF). Kyoto Encyclopedia of Genes and Genome (KEGG) analysis indicated the acquired genes were assigned to 49 KEGG pathways. The majority of these genes were involved in glyoxylate and dicarboxylate metabolism, carbon fixation in photosynthetic organisms, and glycolysis/gluconeogenesis. The interactions between 17K and the 27 proteins with well-documented annotations were verified by conducting yeast two-hybrid assays and 12 of the 27 proteins were verified to interact with 17K. To explore the putative function of the 12 proteins in BYDV-GAV infection, the subcellular localization and expression alterations in the presence of BYDV-GAV were monitored. The results showed that, under the condition of BYDV-GAV infection, RuBisCo, POR, and PPD5 were significantly up-regulated, whereas AEP and CAT1 were significantly down-regulated. Our findings provide insights into the 17K-mediated BYDV-GAV infection process.


17K enhances viral pathogenicity in a heterologous virus expression system. To test whether
17K is associated with viral pathogenicity, the protein was expressed in Nicotiana benthamiana (N. benthamiana) using a PVX-based vector. At 5 days post-infiltration (dpi), the systemic leaves of PVX17K plants showed obvious mosaic symptoms, whereas those treated with PVX showed no symptoms (Fig. 1A). PVX accumulation in PVX17K plants at 5 dpi was indicated by performing Western blotting with PVX CP antibodies, whereas no signal was observed in the PVX control (Figs. 1B, S1). These results suggest that 17K enhances viral pathogenicity of PVX. Additionally, silencing suppressor activity of 17K was tested using a N. benthamiana 16c system, and strong fluorescence was observed at 3 dpi in the combinations GFP + P19 and GFP + 17K, indicating silencing suppressor activity of 17K (Fig. S2).
Subcellular localization pattern of BYDV-GAV 17K. Using PortII (https:// psort. hgc. jp/ form2. html) and seqNLS (http:// mleg. cse. sc. edu/ seqNLS/), 17K was predicted to be localized in the nucleus and would thus show a nuclear localization signal ( Fig. 2A). To illustrate the subcellular localization of BYDV-GAV 17K, 17K-GFP was measured in epidermal cells of N. benthamiana plants. The results showed that the green fluorescent signal of 17K-GFP merged with the red fluorescent signal of H2B-RFP, indicating the nuclear localization of BYDV-GAV 17K (Fig. 2B). Our finding is consistent with a previous study 12 . Notably, we found that 17K was monitored mainly at the nuclear envelope (Fig. 2B).
Identification of 17K-interacting host factors by screening a tobacco library. After screening, a total of 183 yeast clones were collected from the SD/-Trp/-Leu/-His/-Ade (QDO) agar plates. In total, 114 cDNA fragments were obtained by colony PCR amplification and sequentially sequenced. We used BLASTn search, gene ontology (GO), and the Kyoto Encyclopedia of Genes and Genome (KEGG) databases to identify the main functional groups of the acquired clones. Based on the results, a total of 90 genes were annotated. The full list of specific annotation results is shown in Supplementary Tables S1-S3. The identified genes were subjected to a GO enrichment analysis. The 90 acquired genes were classified into 35 functional groups, which belonged to three main categories: biological processes (BP), cellular components (CC), and molecular functions (MF). The main subcategories within BP were cellular processes, metabolic processes, and biological regulation. Within the MF category, catalytic activity and binding were predominant. The three main CC subcategories were cell, cell parts, and organelles (Fig. 3). In addition, 49 KEGG pathways for the acquired genes were identified (Table S3). The pathways with the highest numbers of screened genes were glyoxylate and dicarboxylate metabolism, carbon fixation in photosynthetic organisms, and glycolysis/gluconeogenesis (Fig. 4). Based on the analysis, we found www.nature.com/scientificreports/    (Table S4).

MAQGEQGALAQFGEWLWSNPIEPDQNEELVDAQEEEGQILYLDQQAGLRYSYSQSTTLRP T P Q G Q S S S V P T F R N A Q R F Q V E Y S S P T T V T R S Q T S R L S L S H T R P P L Q S A Q C L L N S T L R
Genes involved in stress responses. Infection by plant viruses induces the expression of a variety of genes that are usually regulated by TFs 16 . TFs are triggers for gene expression and play important roles throughout the lifetime of plants, especially in plant growth, development, and responses to abiotic and biotic stresses 17 . In this study, we identified four TFs after screening, including TF PosF21 (XM_016587072.1), nuclear TF Y subunit C-1-like (XM_009781056.1), zinc finger protein CONSTANS-LIKE 5-like (XM_016580159.1), and GATA TF 5-like (XM_016591336.1). PosF21 shows all the characteristics of a basic region/leucine zipper motif (bZIP) type of DNA-binding domain 18 . As one of the largest TFs, bZIP TFs play pivotal, life-long roles in plant growth 17 .
Genes involved in chloroplast-related functions. Photosynthesis, the most fundamental and complex physiological process in plants, played an important role in viral infection. In this study ten photosynthesis and chloroplast related proteins were identified through screening which are light-harvesting complex I chlorophyll a/b binding protein 3(XM_016643125.1), light-harvesting complex I chlorophyll a/b binding protein 4(XM_016594598.1), light-harvesting complex II chlorophyll a/b binding protein 1(XM_016632344.1), psbP domain-containing protein 5(XM_016648932.1), phosphomethylpyrimidine synthase (XM_016635286.1), glutamine  (Table S5). Among them light-harvesting complex II chlorophyll a/b binding protein, psbP domain-containing protein 5, and glutamine synthetase were previously found to be markedly regulated by BYDV-GAV infection 19 .

Subcellular localization patterns of the identified 17K-interacting host factors.
To determine the subcellular localization of the identified 17K-interacting host factors, we transiently expressed 6 proteins as fusions to the N-terminus of YFP in N. benthamiana leaves. As shown in Fig. 7, POR-YFP and CAT1-YFP showed fluorescence in both the cell periphery and the nucleus. SnRK1-YFP and SnRK2-YFP showed fluorescence in the cell periphery, whereas, BRG2-YFP was perhaps only localized in the nuclear and PPD5 showed the localization at the chloroplast (Figs. 6, S3). The various distributions of identified host proteins suggested 17K protein is possibly involved in several distinct cellular pathways.

Expression of interacting proteins after BYDV infection.
To determine the role of the identified host factors in viral infection, 10 interacting host genes were selected for quantitative RT-PCR (qRT-PCR) analysis using BYDV-inoculated N. benthamiana leaves. The results showed that RuBisCo, POR, and PPD5 were significantly up-regulated by viral infection, whereas AEP and CAT1 were significantly down-regulated (Fig. 7). PPD5 was previously reported to be up-regulated in BYDV-infected wheat leaves at 35 dpi, consistent with our results 19 .

Discussion
Infection by BYDVs causes leaf yellowing and plant dwarfism in wheat, leading to significant economic losses.
To realize successful infection, viruses always have to manipulate or utilize many host factors through directly interacting with them. Multifunctional viral proteins such as γb encoded by Barley stripe mosaic virus (BSMV) which are suppressor of RNA silencing and also affects symptom development and seed transmission of BSMV. Previous studies have shown that multiple host factors interact with γb and affects the roles that γb plays during viral infection [20][21][22] . 17K protein is also a multifunctional protein and its roles in suppression of RNA silencing, virus movement and symptom development are unclear. Identification of interacting host factors will provide  www.nature.com/scientificreports/ insights into the 17 mediated viral process. In this study, we identified 12 host proteins that interact with BYDV 17K. We also tested the possible interaction between 17K and the other BYDV encoded proteins. No interaction was found using Y2H assay. Reactive oxygen species (ROS) such as hydrogen peroxide (H 2 O 2 ) or superoxide anions (O 2 − ) are one of the earliest cellular responses against pathogen infections 20 . Two identified host factors, NbSRC2 and NbCAT1, were involved in ROS production. Arabidopsis SRC2 acts as a novel activator of NADPH oxidase AtRbohF-mediated ROS production and may play a role in cold stress 23 . The rapid production of ROS under biotic or abiotic challenges, known as an oxidative response, is an important signal for both local immune responses and cell-to-cell communication 24 . Catalase serves to protect cells from the toxic effects of hydrogen peroxide. The interaction between 17K and NbSRC2/ NbCAT1 may influence viral infection by regulating ROS production.
Two chloroplast proteins were identified: PsbP domain-containing protein 5 and ribulose bisphosphate carboxylase small-chain S41. Plants contain an extensive family of PsbP domain (PPD) proteins, which are localized in the thylakoid lumen. Members of the PsbP family have been shown to exhibit a variety of functions. Except for the function of photosynthetic electron transfer, the Arabidopsis ppd5 mutant showed striking morphological defects related to a deficiency in strigolactone biosynthesis 25 . RuBisCo is the most abundant enzyme in plants. It catalyzes the carboxylation of ribulose-1,5-biphosphate in chloroplasts and thus is responsible for fixing CO 2 during photosynthesis. Hence, 17K may be involved in different pathways in chloroplasts and thus influence symptom development.
TFs are important players in the response to biotic and abiotic stresses. In this study, two TFs were identified: nuclear TF Y subunit C-1-like (NF-YC1) and zinc finger protein CONSTANS-LIKE 5-like (CO5). Nuclear factor Y (NF-Y) is an evolutionarily conserved trimeric TF complex consisting of three subunits: NF-YA, NF-YB, and NF-YC. Recent studies have shown that the NF-Y complex plays multiple essential roles in plant growth, development, and stress responses 26 . Three NF-YC proteins (NF-YC3, NF-YC4, and NF-YC9) are positive regulators of photomorphogenesis 27 . CONSTANS/CONSTANS-like (CO/COL) belongs to a family of zinc finger TFs and contains one or two B-box zinc finger regions at the N-terminus and a CCT (CO, COL, TOC1) domain at the C-terminus 28 . The CO genes have been reported to be involved in many molecular and genetic processes, including photoperiodically regulated developmental processes and abiotic stresses [29][30][31] . The function of NF-YC1 and CO5 in biotic stress remains unclear. The interaction between 17K and the two TFs may influence the regulation of gene expression by NF-YC1 and CO5, thereby influencing viral accumulation.
Three biotic stress-related genes that interact with 17K were identified, including SNF1-related protein kinase regulatory subunit beta-1-like, SNF1-related protein kinase regulatory subunit beta-2-like, and BOI-related E3 ubiquitin-protein ligase 2.SNF1-related protein kinase 1 (SnRK1) plays a central role in regulating energy and metabolism in plants and has been implicated in responses to abiotic and biotic stresses [32][33][34][35] . It is the bestcharacterized host protein kinase known to be involved in geminivirus infection 34,36,37 . SnRK1 phosphorylation of Rep interferes with viral replication. By contrast, the host defense response is enhanced by SnRK1 phosphorylation of AL2/C2 37 and β-satellite-encoded βC1 protein 36,38 . Protochlorophyllide reductase is a key enzyme in chlorophyll biosynthesis. BOI-related E3 ubiquitin-protein ligases represent a subclass of RING E3 ligases that contribute to plant disease resistance and abiotic stress tolerance through the suppression of pathogen-induced and stress-induced cell death 39 .
The screening and identification of 17K interacting host factor enhances the understanding of the molecular mechanism of BYDV-GAV infection. Since BYDV has different strains, our study will increase the knowledge on the mechanism of infection by BYDV-GAV and other strains.

Materials and methods
Plasmid construction. Primers used for plasmid construction are listed in Table S7. All the available constructs were sequenced.
To construct vectors for yeast two-hybrid analysis, the coding sequence of corresponding genes were amplified and inserted into EcoRI/BamHI digested pGADT7 vectors via homologous recombination.
PVX17K was constructed by introducing 17K into Potato virus X (PVX) vector pGR106 via ClaI and SalI digestion, followed by ligation with T4 DNA ligase (NEB). The fragments used were amplified using primer pairs PVX17KF/PVX17KR. YFP fusion constructs were constructed using gateway strategy. The corresponding genes were cloned into entry vector pDONR221 (Invitrogen) using primer pairs to generate recombination vector. The resultant clone was used to construct the gateway vector 17K-YFP.
Yeast two hybrid assays. The N. tabacum cDNA library was screened according to the protocol handbook provided by the Matchmaker Gold Yeast Two-Hybrid System (Clontech Laboratories, Mountain View, CA, USA). The full-length BYDV-GAV 17K protein was amplified and cloned into yeast vector pGBKT7 to generate the bait vector BD17K. The cDNA library screening and interaction assay were performed as described previously 40 .
Plant materials and virus inoculation. N. benthamiana plants were grown in pots in a growth room under a 16 h light/8 h dark photoperiod at 25 °C with 60% humidity. For agroinfiltration, Agrobacteria strain GV3101 carrying infectious viral clones were suspended in infiltration buffer (10 mM MgCl2, 10 mM MES, and 200 μM acetosyringone, pH 5.6) at an OD 600 of 1, kept at room temperature for 2 to 4 h and infiltrated into N. benthamiana leaves using a 1-mL needleless syringe.