6K2-VPg-Pro polyproteins of monocot-infecting potyviruses phylogenetically separate from that of dicot-infecting potyviruses
To investigate whether the replication site of monocot-infecting potyviruses is same or distinct to that of dicot-infecting potyviruses, we firstly conducted a multiple sequence alignment of potyviral 6K2-VPg-Pro polyprotein amino acid sequences (Supplementary Fig. 1) and performed a phylogenetic analysis (Fig. 1) using the neighbor-joining method implemented with MAFFT online [43]. Multiple sequences alignment showed that the 6K2-VPg-Pro polyproteins share 58.55% identities among the representative 20 potyviruses, and there are 54 absolutely conserved residues for all of them (Supplementary Fig. 1). Strikingly, the 39W, 299N, 413S residues were relatively conserved in dicot-infecting potyviruses, whereas the three sites in monocot-infecting potyviruses were mostly conserved as 39L, 299P, 413D (Supplementary Fig. 1). The phylogenetic tree showed two main clades including a monocot-infecting cluster and a dicot-infecting potyviruses cluster (Fig. 1). In detail, clade 1 contained only monocot-infecting potyviruses including pennisetum mosaic virus (PenMV), maize dwarf mosaic virus (MDMV), SCMV, sorghum mosaic virus (SrMV), johnsongrass mosaic virus (JGMV), and cocksfoot streak virus (CSV), and clade 2 only consists of dicot-infecting potyviruses (Fig. 1).
SCMV replication sites do not correlate with chloroplasts
Double-stranded RNA (dsRNA) is regarded as the hallmark of virus infection [56]. For positive single-stranded RNA viruses, replicative RNA intermediates, namely viral dsRNA is formed during virus replication, whose localization is reasonably thought to be the site of virus replication. To label the replication sites of SCMV in maize cells, we performed the dsRNA binding-dependent fluorescence complementation (dRBFC) assay by co-expressing B2-nYFP and VP35-cYFP in SCMV-infected maize cells following a previous report [36]. In this assay, maize protoplasts isolated from mock- or SCMV-systemically infected leaves (see methods) were transfected with plasmids expressing B2-nYFP and VP35-cYFP. Confocal microscopic analysis showed that fluorescent punctate structures indicating SCMV dsRNA localization were observed in cytoplasm of SCMV-infected maize protoplasts (Fig. 2A), and the reconstituted YFP signals have no obvious association with chloroplasts autofluorescence (Fig. 2A). No fluorescent signal was observed in mock-infected maize protoplasts (Fig. 2A). Western blotting confirmed the infection of SCMV in maize protoplasts (Supplementary Fig. 2A).
Moreover, we performed the dRBFC assay in maize leaves by particle bombardment. The upper maize leaves were collected from healthy and SCMV-systemically infected plants at 9-leaf stage, and subjected to bombardment. The dsRNA-reconstituted YFP fluorescent signals were observed in cytoplasm but did not merge with the red autofluorescence of chloroplasts in SCMV-systemically infected maize leaf cells (Fig. 2B). No YFP fluorescence was observed in healthy leaf cells. Western blotting confirmed the infection of SCMV in maize leaves for bombardment (Supplementary Fig. 2B).
The 6K2-VPg-Pro-induced cytoplasmic vesicles are replication sites of SCMV
Studies on dicot-infecting potyviruses, such as TEV and TuMV, demonstrated that the 6K2-VPg-Pro polyprotein can induce the formation of cytoplasmic vesicles, which were further shown to be their replication sites [8, 26, 35]. To determine whether SCMV-encoded 6K2-VPg-Pro could induce cytoplasmic vesicle structures to function as virus replication sites, we performed the dRBFC assay by co-expressing B2-nYFP and VP35-cYFP with 6K2-VPg-Pro in SCMV-infected maize leaves. To this end, 6K2-VPg-Pro was tagged with mCherry at C-terminal (Fig. 3A). It should be noted that the Glutamine (Q) and Glutamic acid (E) residues preceding the cleavage sites locate at 6K2-VPg and VPg-NIa-Pro junctions were both changed by histidine (H), thereby preventing protein proteolysis [45]. We then conducted a particle bombardment assay in SCMV-infected maize leaves, in which mCherry-tagged 6K2-VPg-Pro (6K2-VPg-Pro-mCherry) was co-expressed with B2-nYFP and VP35-cYFP. The results showed that granular YFP foci formed by dsRNA binding of B2-nYFP and VP35-cYFP perfectly overlapped with the irregular structures induced by 6K2-VPg-Pro-mCherry (Fig. 3B), suggesting that the 6K2-VPg-Pro-induced irregular structures are the active replication sites of SCMV in maize leaf cells.
The 6K2-VPg-Pro-induced replication vesicles reside on multiple intracellular organelles excluding on chloroplasts
Considering that the TuMV 6K2-induced replication vesicles migrate sequentially from ER to chloroplasts for viral genome replication [35], we wondered whether SCMV could also target chloroplasts for replication. For this test, we made a construct 6K2-VPg-Pro-EGFP by fusing EGFP to the C-terminal of 6K2-VPg-Pro (Fig. 4A), and delivered it into protoplasts isolated from SCMV-systemically infected maize leaves. Comparing with the nucleus and cytoplasm-localized wild EGFP, aggregate structures showing green fluorescence could only be observed in cytoplasm of protoplasts transfected with 6K2-VPg-Pro-EGFP (Fig. 4B). Unexpectedly, these structures clearly had no obvious association with the red autofluorescence from chloroplasts (Fig. 4B). Moreover, in N. benthamiana epidermal cells, transient expression of either 6K2-VPg-Pro-EGFP or 6K2-VPg-Pro-mCherry also formed aggregate bodies and punctate structures in cytoplasm (Supplementary Fig. 3). These aggregate bodies and punctate structures did not merge with the chloroplasts autofluorescence (Supplementary Fig. 3), suggesting that SCMV 6K2-VPg-Pro-induced replication vesicles did not reside on chloroplasts.
Previous findings that TuMV 6K2 vesicles accumulate at ER exit sites (ERES) on the ER membrane and subsequently migrate to Golgi apparatus [35, 36] prompted us to investigate whether SCMV 6K2-VPg-Pro-induced vesicles locate at ER and/or Golgi apparatus. We co-expressed 6K2-VPg-Pro-EGFP with an ER marker mCherry-HDEL or a Golgi apparatus marker mCherry-GmMan1 in maize protoplasts [57]. Expectedly, SCMV 6K2-VPg-Pro-EGFP-induced punctate structures colocalized with mCherry-HDEL and mCherry-GmMan1 (Fig. 4C, D), demonstrating that SCMV replicates at both ER and Golgi apparatus. Similar co-localizations were also observed when expressed in N. benthamiana leaf epidermal cells (Fig. 4C, D).
We further tempted to determine whether SCMV 6K2-VPg-Pro could target other organelles such as mitochondria and peroxisomes. Intriguingly, we found that 6K2-VPg-Pro-EGFP-induced punctate structures merged with mCherry-ScCOX4 [57] and peroxisome marker dsRed-SKL [58] in N. benthamiana leaf epidermal cells (Supplementary Fig. 4). Taken together, for replication SCMV can target multiple intracellular organelles including ER, Golgi apparatus, mitochondria and peroxisomes but not chloroplasts.