viruses Article Determination of Protein Interactions among Replication Components of Apple Necrotic Mosaic Virus Zhen-Lu Zhang, Fu-Jun Zhang, Peng-Fei Zheng, Yin-Huan Xie, Chun-Xiang You and Yu-Jin Hao * State Key Laboratory of Crop Biology, College of Horticulture Science and Engineering, Shandong Agricultural University, Tai’an 271000, China; [email protected] (Z.-L.Z.); [email protected] (F.-J.Z.); [email protected] (P.-F.Z.); [email protected] (Y.-H.X.); [email protected] (C.-X.Y.) * Correspondence: [email protected] Received: 16 February 2020; Accepted: 20 April 2020; Published: 22 April 2020 Abstract: Apple mosaic disease is one of the most widely distributed and destructive diseases in apple cultivation worldwide, especially in China, whose apple yields account for more than 50% of the global total. Apple necrotic mosaic virus (ApNMV) is a newly identified ilarvirus that is closely associated with apple mosaic disease in China; however, basic viral protein interactions that play key roles in virus replication and the viral life cycle have not been determined in ApNMV. Here, we first identify an ApNMV–Lw isolate that belongs to subgroup 3 in the genus Ilarvirus. ApNMV–Lw was used to investigate interactions among viral components. ApNMV 1a and 2apol, encoded by RNA1 and RNA2, respectively, were co-localized in plant cell cytoplasm. ApNMV 1a interacted with itself at both the inter- and intramolecular levels, and its N-terminal portion played a key role in these interactions. 1a also interacted with 2apol, and 1a’s C-terminal, together with 2apol’s N-terminal, was required for this interaction. Moreover, the first 115 amino acids of 2apol were sufficient for permitting the 1a–2apol interaction. This study provides insight into the protein interactions among viral replication components of ApNMV, facilitating future investigations on its pathogenicity, as well as the development of strategies to control the virus and disease. Keywords: apple mosaic disease; apple necrotic mosaic virus; virus characterization; protein interaction; protein localization 1. Introduction Apple (Malus domestica) is one of the most important economic fruit trees that grow in many countries around the world, including China. However, fungi, bacteria, viruses, and viroids pose severe threats to the long-term survival of perennial woody plants such as apple trees. Important viruses and viroids of apple trees include apple chlorotic leaf spot virus (ACLSV), apple stem grooving virus (ASGV), apple stem pitting virus (ASPV), and apple scar skin viroid (ASSVd). Virus-induced diseases are one of the most important factors limiting apple production in China [1]. Apple mosaic disease was first reported in Europe in the 1930s and was transmittable by grafting [2]. The typical symptoms of apple mosaic disease only occur on leaves and not on the branches or fruits. The infected leaves usually display pale-yellow irregular spots or chlorosis, amorphous chlorotic bands along or between the veins, and mosaic patterns [3–5]. Currently, virus-induced apple mosaic disease is emerging as a severe threat to apple cultivation and production in many countries. For example, this disease caused reductions in apple yields by 30%–50% and 46% in India [6] and the USA [7], respectively. Photosynthetic deficiencies are the most likely mechanism underlying the yield reductions Viruses 2020, 12, 474; doi:10.3390/v12040474 www.mdpi.com/journal/viruses Viruses 2020, 12, 474 2 of 15 caused by apple mosaic disease, as previous work has shown that the net photosynthetic rate of leaves with mosaic symptoms is decreased to from approximately 3% to more than 45% [8]. In a long period, apple mosaic virus (ApMV), belonging to the genus Ilarvirus in the family Bromoviridae [9], was traditionally believed to be the sole causal agent of apple mosaic disease until prunus necrotic ringspot virus (PNRSV) [10,11], cucumber mosaic virus (CMV) [12], and apple necrotic mosaic virus (ApNMV) [13,14] were also reported to be associated with this disease. Specifically, 90% of the detected apple tree leaves (111 samples collected from different regions of China) with mosaic symptoms were infected with PNRSV, while ApMV was detected in none of them [11]. Furthermore, ApNMV was detected in 92.1% (268 out of 291 apple trees from different regions of China) of symptomatic trees, while ApMV was detected in none of them [14]. Moreover, the distribution of ApNMV was correlated with the symptomatic leaves, and these leaves were unevenly distributed in diseased apple trees [13]. In addition, ApNMV has also been reported to be isolated from crabapple (Malus spp.) leaves with mosaic symptoms [15]. Thus, in China, ApMV is not the only agent that can induce mosaic symptoms, as PNRSV and especially ApNMV, are also associated with apple mosaic disease. However, the induction of mosaic symptoms by ApNMV has not been verified via Koch’s Postulates [16], classical criteria for directly linking a specific pathogen with its corresponding symptom. ApNMV was first reported to be associated with the apple mosaic disease by a Japanese group [13]. ApNMV, along with ApMV and PNRSV, is classified into subgroup 3 in the genus Ilarvirus in the family Bromoviridae [13]. Similar to other ilarviruses, ApNMV contains three positive single-strand genomic RNA segments (RNA1, RNA2, and RNA3) and a subgenomic RNA4 that derived from RNA3 [13,14]. The protein encoded by RNA1 contains an N-terminal methyltransferase (MET) domain and a C-terminal NTP-binding helicase (HEL) domain. RNA2 encodes the viral RNA-dependent RNA polymerase (RdRp), and both of the proteins encoded by RNA1 and RNA2 are responsible for viral replication [13]. RNA3 encodes the movement protein (MP), while the coat protein (CP) is encoded by a subgenomic RNA4. MP and CP are involved in the viral movement, including cell-to-cell movement and systematic transportation [13]. Here, we obtained the full genome sequences of ApNMV–Lw isolated from an orchard in Laiwu, China. The replication proteins of ApNMV are co-localized in the cytoplasm in plant epidermal cells. Meanwhile, ApNMV 1a interacts with itself, and the N-terminal MET domain plays a key role in 1a’s inter- and intra-molecular interactions. In addition, 1a’s C-terminal HEL portion interacts with the N-terminal of 2apol. The first 115 amino acids (aa) of 2apol is sufficient for supporting the 1a–2apol interaction. 2. Materials and Methods 2.1. Plant Materials and Growth Conditions Leaves with mosaic symptoms were collected from Fuji apple trees in an orchard in Laiwu, Shandong Province, China. Wild-type N. benthamiana were maintained in a greenhouse with a 16:8 h light:dark at 23 ◦C 2.2. Plasmid Construction and Genetic Transformation All PCR products were first cloned into pEASY-Blunt Simple cloning vector (Transgen), and then were constructed to expression vectors. The 1a–GFP and 2apol–GFP constructs were generated using the modified pRI-101 vector [17]. cLuc–1a, 1a–nLuc, cLuc–2apol, and 2apol–nLuc were obtained by inserting 1a and 2apol into pGreenII 62–SK–nLuc and –cLuc, respectively [18]. For the BiFC assay, 1a was inserted into 35S::SPYCE–cYFP or 35S::SPYNE–nYFP to construct 1a–cYFP and 1a–nYFP, respectively [19]. 1a and 2apol were inserted into pGEX–4T–1 and pET–32a to obtain the GST- and HIS-tagged fusion proteins, respectively, for the pull-down assay. pGAD424 and pGBT9 were used to make the constructs in the Y2H experiments. The primers used to amplify the fragments and make Viruses 2020, 12, 474 3 of 15 constructs are shown in Table S1. Agrobacterium strain LBA4404 was used for agro-infiltration to achieve the transformation. 2.3. RNA Extraction and Complete Genomic RNA Sequence Acquisition Total RNAs were extracted using the hot phenol method [20]. Briefly, the ground samples were treated with hot phenol and extracted with phenol/chloroform solution. Total RNAs were then precipitated by sodium acetate and washed with 70% ethanol. Lastly, RNAs were resuspended in RNase-free water. First-strand cDNAs were synthesized with the PrimeScript RT reagent Kit (TaKaRa BIO INC, Otsu, Shiga, Japan) following the manufacturer’s instructions and using Random 6 as a primer. The middle fragments of RNA1, RNA2, and RNA3 were amplified by RNA1–F/R, RNA2–F/R, and RNA3–F/R primer pairs, respectively. The flanked 50 and 30 ends of each genomic RNA segment were amplified using the SMARTer RACE 50/30 Kit (Clontech Laboratories Inc, Mountain view, CA, USA) following the user manual. Finally, the three amplified fragments (50 end, middle fragment, and 30 end) were aligned and assembled to obtain the full length of each RNA segment, and, in turn, the full genome sequences of the ApNMV–Lw isolate. All of the primers used in genome RNA amplification are shown in Supplemental Table S2. 2.4. Homology Comparison and Phylogenetic Analysis The similarity of ApNMV–Lw with other ilarviruses at the nucleotides levels of RNA1, RNA2, and RNA3 as well as the amino acid levels of 1a, 2apol, MP, and CP were analyzed by DNAman (Lynnon Biosoft, San Ramon, CA, USA) software. Phylogenetic trees were constructed with MEGA (version 7.0.14, developed by Sudhir Kumar-the prject leader-from the Arizona State University, USA) software by using ClustalW and the neighbor-joining method [21] with 1000 bootstrap replications based on the nucleotide sequences of RNA1, RNA2, and RNA3 of ApNMV–Lw and other ilarviruses. 2.5. Yeast-Two-Hybrid Assay The Y2H assay was conducted as previously described [22]. Briefly, the cDNAs of the target genes were amplified and inserted into the pGAD424 and pGBT9 vectors. The bait and prey constructs were transformed into the Y2H Gold yeast strain and grown on the selection medium without lucine (Leu) and tryptophan (Trp). The yeast colonies were then grown on SD base-Leu/-Trp medium and were transferred to selection medium without Leu, Trp, histidine, and adenine to characterize the interactions between bait and prey proteins.