In plants, autophagy is involved in responses to viral infection. However, the role of host factors in mediating autophagy to suppress viruses is poorly understood. A previously uncharacterized plant protein, NbP3IP, was shown to interact with p3, an RNA-silencing suppressor protein encoded by Rice stripe virus (RSV), a negative-strand RNA virus. The potential roles of NbP3IP in RSV infection were examined. NbP3IP degraded p3 through the autophagy pathway, thereby affecting the silencing suppression activity of p3. Transgenic overexpression of NbP3IP conferred resistance to RSV infection in Nicotiana benthamiana. RSV infection was promoted in ATG5-or ATG7-silenced plants and was inhibited in GAPC-silenced plants where autophagy was activated, confirming the role of autophagy in suppressing RSV infection. NbP3IP interacted with NbATG8f, indicating a potential selective autophagosomal cargo receptor role for P3IP. Additionally, the rice NbP3IP homolog (OsP3IP) also mediated p3 degradation and interacted with OsATG8b and p3. Through identification of the involvement of P3IP in the autophagy-mediated degradation of RSV p3, we reveal a new mechanism to antagonize the infection of RSV, and thereby provide the first evidence that autophagy can play an antiviral role against negative-strand RNA viruses.

The chloroplast protein ferredoxin 1 (FD1), with roles in the chloroplast electron transport chain, is known to interact with the coat proteins (CPs) of Tomato mosaic virus and Cucumber mosaic virus. However, our understanding of the roles of FD1 in virus infection remains limited. Here, we report that the Potato virus X (PVX) p25 protein interacts with FD1, whose mRNA and protein levels are reduced by PVX infection or by transient expression of p25. Silencing of FD1 by Tobacco rattle virus-based virus-induced gene silencing (VIGS) promoted the local and systemic infection of plants by PVX. Use of a drop-and-see (DANS) assay and callose staining revealed that the permeability of plasmodesmata (PDs) was increased in FD1-silenced plants together with a consistently reduced level of PD callose deposition. After FD1 silencing, quantitative reverse transcription–real-time PCR (qRT–PCR) analysis and LC-MS revealed these plants to have a low accumulation of the phytohormones abscisic acid (ABA) and salicylic acid (SA), which contributed to the decreased callose deposition at PDs. Overexpression of FD1 in transgenic plants manifested resistance to PVX infection, but the contents of ABA and SA, and the PD callose deposition were not increased in transgenic plants. Overexpression of FD1 interfered with the RNA silencing suppressor function of p25. These results demonstrate that interfering with FD1 function causes abnormal plant hormone-mediated antiviral processes and thus enhances PVX infection.

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Rice stripe virus (RSV) causes one of the most serious viral diseases of rice. RNA interference is one of the most efficient ways to control viral disease. In this study, we constructed an amiRNA targeting the RSV MP gene (amiR MP) based on the backbone sequence of the osa-MIR528 precursor, and obtained marker-free transgenic rice plants constitutively expressing amiR MP by Agrobacterium tumefaciens-mediated transformation. A transient expression assay demonstrated that dimeric amiR MP could be effectively recognized and cleaved at the target MP gene in plants. Northern blot of miRNA indicated that amiR MP-mediated viral resistance could be stably inherited. The transgenic rice plants were highly resistant to RSV (73–90%). Our research provides novel rice germplasm for RSV control.

30In plants, autophagy is involved in responses to viral infection. However, understanding 31 of new host factors mediating autophagic clearance of plant viruses is very limited. We 32 here identified a new host factor NbP3IP participating in autophagy-mediated plant 33 defense against viral infection. NbP3IP interacted with p3, a RNA silencing suppressor 34 encoded by Rice stripe virus (RSV), a negative-strand RNA virus, and mediated its 35 autophagic degradation. NbP3IP could also interact with NbATG8f, which was required 36 for NbP3IP-miediated p3 degradation. Overexpression of NbP3IP induced autophagy and 37 down-regulation of NbP3IP reduced autophagy. Both overexpression of NbP3IP and 38 silencing of GAPC, which also induces autophagy, inhibited RSV infection. In contrast, 39 silencing of ATG7 promoted RSV infection. Thus, through identification of a new 40 potential selective autophagy receptor P3IP, we revealed a new mechanism of 41 autophagy-mediated plant defense against plant viruses and provided the first evidence 42 that plant autophagy can also play an antiviral role against negative-strand RNA viruses. 43 KEY WORDS 44 Autophagy; Rice stripe virus; p3; NbP3IP; ATG8 45 46 Rice stripe virus (RSV), transmitted by the small brown planthopper (SBPH; Laodelphax 48 striatellus Fallén), causes serious epidemics in East Asia, including China, Japan and 49 Korea (Cheng et al., 2008). RSV belongs to the genus Tenuivirus and only infects plants 50 of the family Poaceae by natural SBPH transmission, which is a barrier to studies 51exploring the pathogenesis of RSV and its interaction with plants in the field or with its 52 3 insect vector. In the laboratory, however, RSV can infect the experimental plant Nicotiana 53 benthamiana by mechanical inoculation, which has been adopted as a very useful model 54 system for studying RSV-plant interactions (Xiong et al., 2008; Yuan et al., 2011; Zhang 55 et al., 2012; Kong et al., 2013; Fu et al., 2018). RSV has four single-stranded RNA 56 genome segments. RNA1 (~9 kb) is negative-sense and has a single open reading frame 57 (ORF), encoding the RNA-dependent RNA polymerase (RdRP), in its complementary 58 strand. Each of the other three segments (RNA2, 3.5 kb; RNA3, 2.5 kb; RNA4, 2.2 kb) 59 are ambisense and contain two non-overlapping ORFs on opposite strands, separated by a 60 non-coding intergenic region (IR) that functions in termination of transcription (Zhu et al., 61 1991; Zhu et al.

Previously we reported that the multifunctional cylindrical inclusion (CI) protein of turnip mosaic virus (TuMV) is targeted to endosomes through the interaction with the medium subunit of adaptor protein complex 2 (AP2β), which is essential for viral infection. Although several functionally important regions in the CI have been identified, little is known about the determinant(s) for endosomal trafficking. The CI protein contains seven conserved acidic dileucine motifs [(D/E)XXXL(L/I)] typical of endocytic sorting signals recognized by AP2β. Here, we selected five motifs for further study and identified that they all were located in the regions of CI interacting with AP2β. Coimmunoprecipitation assays revealed that alanine substitutions in the each of these acidic dileucine motifs decreased binding with AP2β. Moreover, these CI mutants also showed decreased accumulation of punctate bodies, which enter endocytic‐tracking styryl‐stained endosomes. The mutations were then introduced into a full‐length infectious clone of TuMV, and each mutant had reduced viral replication and systemic infection. The data suggest that the acidic dileucine motifs in CI are indispensable for interacting with AP2β for efficient viral replication. This study provides new insights into the role of endocytic sorting motifs in the intracellular movement of viral proteins for replication.

Quantitative real-time PCR (RT-qPCR) is a widely used method for studying alterations in gene expression upon infections caused by diverse pathogens such as viruses. Positive-sense single-stranded (ss(+)) RNA viruses form a major part of all known plant viruses, and some of them are damaging pathogens of agriculturally important crops. Analysis of gene expression following infection by ss(+) RNA viruses is crucial for the identification of potential anti-viral factors. However, viral infections are known to globally affect gene expression and therefore selection and validation of reference genes for RT-qPCR is particularly important. In this study, the expression of commonly used reference genes for RT-qPCR was studied in Nicotiana benthamiana following single infection by 11 ss(+) RNA viruses, including five tobamoviruses, four potyviruses, one potexvirus and one polerovirus. Stability of gene expression was analyzed in parallel by four commonly used algorithms: geNorm, NormFinder, BestKeeper, and Delta CT, and RefFinder was finally used to summarize all the data. The most stably expressed reference genes differed significantly among the viruses, even when those viruses were from the same genus. Our study highlights the importance of the selection and validation of reference genes upon different viral infections.

There are four ALY proteins in Arabidopsis thaliana, among which ALY1 has been identified to transport the mRNA of the RNA-directed DNA methylation (RdDM) factor ARGONAUTE6 out of the nucleus (Choudury et al., 2019). In mammalian cells, Aly/REF is involved in aiding nuclear export of viral RNA by directly interacting with viral proteins (Balasubramaniam et al., 2013; Tian et al., 2013). In plants, tomato bushy stunt virus (TBSV) P19 can interact with ALY