Ubiquitin like protein 5 (UBL5) interacts with other proteins to regulate their function but differs from ubiquitin and other UBLs because it does not form covalent conjugates. Ubiquitin and most UBLs mediate the degradation of target proteins through the 26S proteasome but it is not known if UBL5 can also do that. Here we found that the UBL5s of rice and Nicotiana benthamiana interacted with rice stripe virus (RSV) p3 protein. Silencing of NbUBL5s in N. benthamiana facilitated RSV infection, while UBL5 overexpression conferred resistance to RSV in both N. benthamiana and rice. Further analysis showed that NbUBL5.1 impaired the function of p3 as a suppressor of silencing by degrading it through the 26S proteasome. NbUBL5.1 and OsUBL5 interacted with RPN10 and RPN13, the receptors of ubiquitin in the 26S proteasome. Furthermore, silencing of NbRPN10 or NbRPN13 compromised the degradation of p3 mediated by NbUBL5.1. Together, the results suggest that UBL5 mediates the degradation of RSV p3 protein through the 26S proteasome, a previously unreported plant defense strategy against RSV infection. The ubiquitin-mediated 26S proteasome has been reported to inhibit viral infection in plants. We here found that the 26S proteasome also degraded the p3 suppressor of RNA silencing of rice stripe virus to inhibit viral infection, and that the degradation was mediated by ubiquitin-like protein 5 (UBL5) but not by ubiquitin. UBL5 has been reported to modulate the proteins with which it interacts by being part of a functional complex, but not by directly mediating its degradation. Our results suggest that UBL5 may also play a role in mediating target protein degradation, a previously unreported function and one that it shares with ubiquitin. This is also the first report that UBL5 interacts with a pathogen protein and that UBL5-mediated regulation might also participate in plant defense against pathogens.

Jasmonic acid (JA) is a crucial hormone in plant antiviral immunity. Increasing evidence shows that viruses counter this host immune response by interfering with JA biosynthesis and signaling. However, the mechanism by which viruses affect JA biosynthesis is still largely unexplored. Here, we show that a highly conserved chloroplast protein cpSRP54 was downregulated in Nicotiana benthamiana infected by turnip mosaic virus (TuMV). Its silencing facilitated TuMV infection. Furthermore, cpSRP54 interacted with allene oxide cyclases (AOCs), key JA biosynthesis enzymes, and was responsible for delivering AOCs onto the thylakoid membrane (TM). Interestingly, TuMV P1 protein interacted with cpSRP54 and mediated its degradation via the 26S proteosome and autophagy pathways. The results suggest that TuMV has evolved a strategy, through the inhibition of cpSRP54 and its delivery of AOCs to the TM, to suppress JA biosynthesis and enhance viral infection. Interaction between cpSRP54 and AOCs was shown to be conserved in Arabidopsis and rice, while cpSRP54 also interacted with, and was degraded by, pepper mild mottle virus (PMMoV) 126 kDa protein and potato virus X (PVX) p25 protein, indicating that suppression of cpSRP54 may be a common mechanism used by viruses to counter the antiviral JA pathway.

Autophagy is induced by viral infection and has antiviral functions in plants, but the underlying mechanism is poorly understood. We previously identified a viral small interfering RNA (vsiRNA) derived from rice stripe virus (RSV) RNA4 that contributes to the leaf-twisting and stunting symptoms caused by this virus by targeting the host eukaryotic translation initiation factor 4A (eIF4A) mRNA for silencing. In addition, autophagy plays antiviral roles by degrading RSV p3 protein, a suppressor of RNA silencing. Here, we demonstrate that eIF4A acts as a negative regulator of autophagy in Nicotiana benthamiana. Silencing of NbeIF4A activated autophagy and inhibited RSV infection by facilitating autophagic degradation of p3. Further analysis showed that NbeIF4A interacts with NbATG5 and interferes with its interaction with ATG12. Overexpression of NbeIF4A suppressed NbATG5-activated autophagy. Moreover, expression of vsiRNA-4A, which targets NbeIF4A mRNA for cleavage, induced autophagy by silencing NbeIF4A. Finally, we demonstrate that eIF4A from rice, the natural host of RSV, also interacts with OsATG5 and suppresses OsATG5-activated autophagy, pointing to the conserved function of eIF4A as a negative regulator of antiviral autophagy. Taken together, these results reveal that eIF4A negatively regulates antiviral autophagy by interacting with ATG5 and that its mRNA is recognized by a virus-derived siRNA, resulting in its silencing, which induces autophagy against viral infection.

Rice stripe virus (RSV) is one of the most devastating viral pathogens in rice and can also cause the general chlorosis symptom in Nicotiana benthamiana plants. The chloroplast changes associated with chlorosis symptom suggest that RSV interrupts normal chloroplast functions. Although the change of proteins of the whole cell or inside the chloroplast in response to RSV infection have been revealed by proteomics, the mechanisms resulted in chloroplast-related symptoms and the crucial factors remain to be elucidated. RSV infection caused the malformation of chloroplast structure and a global reduction of chloroplast membrane protein complexes in N. benthamiana plants. Here, both the protoplast proteome and the chloroplast proteome were acquired simultaneously upon RSV infection, and the proteins in each fraction were analyzed. In the protoplasts, 1128 proteins were identified, among which 494 proteins presented significant changes during RSV; meanwhile, 659 proteins were identified from the chloroplasts, and 279 of these chloroplast proteins presented significant change. According to the label-free LC–MS/MS data, 66 nucleus-encoded chloroplast-related proteins (ChRPs), which only reduced in chloroplast but not in the whole protoplast, were identified, indicating that these nuclear-encoded ChRPswere not transported to chloroplasts during RSV infection. Gene ontology (GO) enrichment analysis confirmed that RSV infection changed the biological process of protein targeting to chloroplast, where 3 crucial ChRPs (K4CSN4, K4CR23, and K4BXN9) were involved in the regulation of protein targeting into chloroplast. In addition to these 3 proteins, 41 among the 63 candidate proteins were characterized to have chloroplast transit peptides. These results indicated that RSV infection changed the biological process of protein targeting into chloroplast and the location of ChRPs through crucial protein factors, which illuminated a new layer of RSV–host interaction that might contribute to the symptom development.

Pepper vein yellows viruses (PeVYV) are phloem-restricted viruses in the genus Polerovirus, family Luteoviridae. Typical viral symptoms of PeVYV including interveinal yellowing of leaves and upward leaf curling were observed in pod pepper plants (Capsicum frutescens) growing in Wenshan city, Yunnan province, China. The complete genome sequence of a virus from a sample of these plants was determined by next-generation sequencing and RT-PCR. Pod pepper vein yellows virus (PoPeVYV) (MT188667) has a genome of 6015 nucleotides, and the characteristic genome organization of a member of the genus Polerovirus. In the 5′ half of its genome (encoding P0 to P4), PoPeVYV is most similar (93.1% nt identity) to PeVYV-3 (Pepper vein yellows virus 3) (KP326573) but diverges greatly in the 3′-part encoding P5, where it is most similar (91.7% nt identity) to tobacco vein distorting virus (TVDV, EF529624) suggesting a recombinant origin. Recombination analysis predicted a single recombination event affecting nucleotide positions 4126 to 5192 nt, with PeVYV-3 as the major parent but with the region 4126–5192 nt derived from TVDV as the minor parent. A full-length clone of PoPeVYV was constructed and shown to be infectious in C. frutescens by RT-PCR and the presence of icosahedral viral particles.