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Positive-sense single-stranded RNA (+ssRNA) viruses, the most abundant viruses of eukaryotes in nature, require the synthesis of negative-sense RNA (−RNA) using their genomic (positive-sense) RNA (+RNA) as a template for replication. Based on current evidence, viral proteins are translated via viral +RNAs, whereas −RNA is considered to be a viral replication intermediate without coding capacity. Here, we report that plant and animal +ssRNA viruses contain small open reading frames (ORFs) in their −RNA (reverse ORFs [rORFs]). Using turnip mosaic virus (TuMV) as a model for plant +ssRNA viruses, we demonstrate that small proteins encoded by rORFs display specific subcellular localizations, and confirm the presence of rORF2 in infected cells through mass spectrometry analysis. The protein encoded by TuMV rORF2 forms punctuate granules that are localized in the perinuclear region and co-localized with viral replication complexes. The rORF2 protein can directly interact with the viral RNA-dependent RNA polymerase, and mutation of rORF2 completely abolishes virus infection, whereas ectopic expression of rORF2 rescues the mutant virus. Furthermore, we show that several rORFs in the −RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have the ability to suppress type I interferon production and facilitate the infection of vesicular stomatitis virus. In addition, we provide evidence that TuMV might utilize internal ribosome entry sites to translate these small rORFs. Taken together, these findings indicate that the −RNA of +ssRNA viruses can also have the coding capacity and that small proteins encoded therein play critical roles in viral infection, revealing a viral proteome larger than previously thought. It is generally accepted that the negative-sense RNA (−RNA) of positive-sense single-stranded RNA (+ssRNA) viruses lacks coding capacity. This study shows that plant and animal +ssRNA viruses contain small open reading frames (ORFs) in their −RNA (rORFs) that display specific subcellular localizations and are important for viral infection. These small rORFs might be translated using internal ribosome entry sites as a translational strategy. [ABSTRACT FROM AUTHOR]

RNA quality control nonsense-mediated decay is involved in viral restriction in both plants and animals. However, it is not known whether two other RNA quality control pathways, nonstop decay and no-go decay, are capable of restricting viruses in plants. Here, we show that the evolutionarily conserved Pelota–Hbs1 complex negatively regulates infection of plant viruses in the family Potyviridae (termed potyvirids), the largest group of plant RNA viruses that accounts for more than half of the viral crop damage worldwide. Pelota enables the recognition of the functional G 1-2 A 6-7 motif in the P3 cistron, which is conserved in almost all potyvirids. This allows Pelota to target the virus and act as a viral restriction factor. Furthermore, Pelota interacts with the SUMO E2-conjugating enzyme SCE1 and is SUMOylated in planta. Blocking Pelota SUMOylation disrupts the ability to recruit Hbs1 and inhibits viral RNA degradation. These findings reveal the functional importance of Pelota SUMOylation during the infection of potyvirids in plants. This study reveals that the Pelota–Hbs1 RNA surveillance complex restricts the infection of potyvirids. Pelota recognizes the conserved G 1-2 A 6-7 motif in potyvirids for viral RNA decay. SUMOylation of Pelota is required for mediation of Pelota–Hbs1 complex functions in antiviral restriction. [ABSTRACT FROM AUTHOR]

Recently reported adenine base editors (ABEs) exhibit powerful potential for targeted gene correction as well as developing gain-of-function mutants and novel germplasms for both gene function studies and crop breeding. However, editing efficiency varies significantly among different target sites. Here, we investigated the activities of three evolved E. coli adenosine deaminase TadA variants (TadA8e, TadA8.17, and TadA8.20) side-by-side in transgenic rice. We found that TadA8e outperforms TadA8.17 and TadA8.20, and induces efficient A-to-G conversion at all tested sites in the rice genome, including those that were uneditable by ABE7.10 in our previous experiments. Furthermore, V82S/Q154R mutations were incorporated into TadA8e, resulting in a new variant that we named TadA9. Our data show that TadA9 is broadly compatible with CRISPR/SpCas9, CRISPR/SpCas9-NG, and CRISPR/SpRY, as well as CRISPR/ScCas9 nickase systems, achieving comparable or enhanced editing in a larger editing window at diverse PAM sites as compared with TadA8e. Finally, TadA9 was used to simultaneously install novel SNPs in four endogenous herbicide target genes in the commercial rice cultivar Nangeng 46 for potential field application in weed control. Collectively, we successfully generated a series of novel ABEs that can efficiently edit adenosines in the rice genome. Our findings suggest that TadA9 and TadA8e have great potentials in the development of plant base editors and crop molecular breeding. This study reveals that TadA8e outperforms TadA8.17 and TadA8.20 and achieves efficient adenine base editing in rice, whileTadA9 achieves comparable or enhanced editing in a larger editing window at tested sites as compared with TadA8e and can simultaneously edits four endogenous target genes at high efficiency. Both TadA9 and TadA8e are broadly compatible with CRISPR/SpCas9, CRISPR/SpCas9-NG, and CRISPR/SpRY, as well as CRISPR/ScCas9 nickase systems for efficient adenine base editing by targeting diverse PAM sequences in rice.. [ABSTRACT FROM AUTHOR]

Recently developed CRISPR-mediated base editors, which enable the generation of numerous nucleotide changes in target genomic regions, have been widely adopted for gene correction and generation of crop germplasms containing important gain-of-function genetic variations. However, to engineer target genes with unknown functional SNPs remains challenging. To address this issue, we present here a base-editing-mediated gene evolution (BEMGE) method, employing both Cas9n-based cytosine and adenine base editors as well as a single-guide RNA (sgRNA) library tiling the full-length coding region, for developing novel rice germplasms with mutations in any endogenous gene. To this end, OsALS1 was artificially evolved in rice cells using BEMGE through both Agrobacterium -mediated and particle-bombardment-mediated transformation. Four different types of amino acid substitutions in the evolved OsALS1, derived from two sites that have never been targeted by natural or human selection during rice domestication, were identified, conferring varying levels of tolerance to the herbicide bispyribac-sodium. Furthermore, the P171F substitution identified in a strong OsALS1 allele was quickly introduced into the commercial rice cultivar Nangeng 46 through precise base editing with the corresponding base editor and sgRNA. Collectively, these data indicate great potential of BEMGE in creating important genetic variants of target genes for crop improvement. Base-editing-mediated gene evolution (BEMGE) efficiently drives artificial evolution of target genes in planta , generating a large number of novel alleles in rice in a short time. The P171F substitution in OsALS1 renders rice plants resistant to the herbicide bispyribac-sodium. Important verified single-nucleotide polymorphism can be rapidly introduced into elite rice cultivars through precise base editing. BEMGE is of great value in the generation of important genetic variants of target genes for crop improvement. [ABSTRACT FROM AUTHOR]

CRISPR technologies enabling precise genome manipulation are valuable for gene function studies and molecular crop breeding. However, the requirement of a protospacer adjacent motif (PAM), such as NGG and TTN, for Cas protein recognition restricts the selection of targetable genomic loci in practical applications of CRISPR technologies. Recently Cas9-NG, which recognizes a minimal NG PAM, was reported to expand the targeting space of genome editing in human cells, but it remains unclear whether this Cas9 variant can be used in plants. In this study, we evaluated the nuclease activity of Cas9-NG toward various NGN PAMs by targeting endogenous genes in transgenic rice. We found that Cas9-NG edits all NGG, NGA, NGT, and NGC sites with impaired activity, while the gene-edited plants were dominated by monoallelic mutations. Cas9-NG-engineered base editors were then developed and used to generate OsBZR1 gain-of-function plants that can not be created by other available Cas9-engineered base editors. Moreover, we showed that a Cas9-NG-based transcriptional activator efficiently upregulated the expression of endogenous target genes in rice. In addition, we discovered that Cas9-NG recognizes NAC, NTG, NTT, and NCG apart from NG PAM. Together, these findings demonstrate that Cas9-NG can greatly expand the targeting scope of genome-editing tools, showing great potential for targeted genome editing, base editing, and genome regulation in plants. Cas9-NG endonuclease recognizes NG, NAC, NTG, NTT and NCG PAM, inducing target gene mutations in rice. Cas9-NG nickase is compatible with nucleoside deaminases and transcriptional activation domain, functioning in base editing and transcriptional regulation of target gene in rice. [ABSTRACT FROM AUTHOR]

Abstract Many geminivirus C4 proteins induce severe developmental abnormalities in plants. We previously demonstrated that Tomato leaf curl Yunnan virus (TLCYnV) C4 induces plant developmental abnormalities at least partically by decreasing the accumulation of NbSKη, an ortholog of Arabidopsis BIN2 kinase involved in the brassinosteroid signaling pathway, in the nucleus through directing it to the plasma membrane. However, the molecular mechanism by which the membrane-associated C4 modifies the localization of NbSKη in the host cell remains unclear. Here, we show that TLCYnV C4 is a nucleocytoplasmic shuttle protein, and that C4 shuttling is accompanied by nuclear export of NbSKη. TLCYnV C4 is phosphorylated by NbSKη in the nucleus, which promotes myristoylation of the viral protein. Myristoylation of phosphorylated C4 favors its interaction with exportin-α (XPO I), which in turn facilitates nuclear export of the C4/NbSKη complex. Supporting this model, chemical inhibition of N -myristoyltransferases or exportin-α enhanced nuclear retention of C4, and mutations of the putative phosphorylation or myristoylation sites in C4 resulted in increased nuclear retention of C4 and thus decreased severity of C4-induced developmental abnormalities. The impact of C4 on development is also lessened when a nuclear localization signal or a nuclear export signal is added to its C-terminus, restricting it to a specific cellular niche and therefore impairing nucleocytoplasmic shuttling. Taken together, our results suggest that nucleocytoplasmic shuttling of TLCYnV C4, enabled by phosphorylation by NbSKη, myristoylation, and interaction with exportin-α, is critical for its function as a pathogenicity factor. TLCYnV C4 induces severe developmental abnormalities by decreasing the accumulation of NbSKη in the nucleus. This study provides evidence that TLCYnV C4 is phosphorylated by NbSKη in the nucleus, and that phosphorylation promotes myristoylation. Myristoylation of phosphorylated C4 enables its interaction with exportin-α to mediate nuclear export of the C4/NbSKη complex and its relocalization to the plasma membrane. [ABSTRACT FROM AUTHOR]

Abstract Remorins are plant-specific membrane-associated proteins and were proposed to play crucial roles in plant–pathogen interactions. However, little is known about how pathogens counter remorin-mediated host responses. In this study, by quantitative whole-proteome analysis we found that the remorin protein (NbREM1) is downregulated early in Rice stripe virus (RSV) infection. We further discovered that the turnover of NbREM1 is regulated by S-acylation modification and its degradation is mediated mainly through the autophagy pathway. Interestingly, RSV can interfere with the S-acylation of NbREM1, which is required to negatively regulate RSV infection by restricting virus cell-to-cell trafficking. The disruption of NbREM1 S-acylation affects its targeting to the plasma membrane microdomain, and the resulting accumulation of non-targeted NbREM1 is subjected to autophagic degradation, causing downregulation of NbREM1. Moreover, we found that RSV-encoded movement protein, NSvc4, alone can interfere with NbREM1 S-acylation through binding with the C-terminal domain of NbREM1 the S-acylation of OsREM1.4, the homologous remorin of NbREM1, and thus remorin-mediated defense against RSV in rice, the original host of RSV, indicating that downregulation of the remorin protein level by interfering with its S-acylation is a common strategy adopted by RSV to overcome remorin-mediated inhibition of virus movement. Fu et al. determine a plant remorin protein acting as a negative regulator of Rice stripe virus infection by reducing viral cell-to-cell movement. To counter this defensive response, virus encodes the NSvc4 protein to interfere with S-acylation of remorin and induce its autophagic degradation. [ABSTRACT FROM AUTHOR]

The βC1 protein encoded by the Tomato yellow leaf curl China virus -associated betasatellite functions as a pathogenicity determinant. To better understand the molecular basis whereby βC1 functions in pathogenicity, a yeast two-hybrid screen of a tobacco cDNA library was carried out using βC1 as the bait. The screen revealed that βC1 interacts with a tobacco RING-finger protein designated NtRFP1, which was further confirmed by the bimolecular fluorescence complementation and co-immunoprecipitation assays in Nicotiana benthamiana cells. Expression of NtRFP1 was induced by βC1, and in vitro ubiquitination assays showed that NtRFP1 is a functional E3 ubiquitin ligase that mediates βC1 ubiquitination. In addition, βC1 was shown to be ubiquitinated in vivo and degraded by the plant 26S proteasome. After viral infection, plants overexpressing NtRFP1 developed attenuated symptoms, whereas plants with silenced expression of NtRFP1 showed severe symptoms. Other lines of evidence showed that NtRFP1 attenuates βC1-induced symptoms through promoting its degradation by the 26S proteasome. Taken together, our results suggest that tobacco RING E3 ligase NtRFP1 attenuates disease symptoms by interacting with βC1 to mediate its ubiquitination and degradation via the ubiquitin/26S proteasome system. [ABSTRACT FROM AUTHOR]

An abstract of the study "A Layered Defense Strategy Mediated by Rice E3 Ubiquitin Ligases against Diverse Pathogens," by Xuetao Shi and colleagues is presented.

Summary: In plants, DNA methylation can be mediated by a class of Argonaute4 (AGO4)-associated heterochromatic siRNAs (hc-siRNAs), through a pathway termed RNA-directed DNA methylation (RdDM). It has been thought that RdDM is solely a nuclear process, as both the biogenesis and functioning of hc-siRNAs take place in the nucleus. In this study, we unexpectedly found that hc-siRNAs are predominantly present in the cytoplasm. We demonstrated that AGO4 is loaded with hc-siRNAs in the cytoplasm and the formation of mature AGO4/siRNA complexes requires HSP90 and the cleavage activity of AGO4. Intriguingly, siRNA binding facilitates the redistribution of AGO4 into the nucleus, likely through inducing conformational change that leads to the exposure of the nuclear localization signal (NLS). Our findings reveal an unsuspected cytoplasmic step in the RdDM pathway. We propose that selective nuclear import of mature AGO4/siRNA complexes is a key regulatory point prior to the effector stage of RdDM. [ABSTRACT FROM AUTHOR]

Rice stripe virus (RSV) causes severe diseases in Oryza sativa (rice) in many Eastern Asian countries. Disease-specific protein (SP) of RSV is a non-structural protein and its accumulation level in rice plant was shown to determine the severity of RSV symptoms. Here, we present evidence that expression of RSV SP alone in rice or Nicotiana benthamiana did not produce visible symptoms. Expression of SP in these two plants, however, enhanced RSV- or Potato virus X (PVX)-induced symptoms. Through yeast two-hybrid screening, GST pull-down, and bimolecular fluorescence complementation assays, we demonstrated that RSV SP interacted with PsbP, a 23-kDa oxygen-evolving complex protein, in both rice and N. benthamiana. Furthermore, our investigation showed that silencing of PsbP expression in both plants increased disease symptom severity and virus accumulation. Confocal microscopy using N. benthamiana protoplast showed that PsbP accumulated predominantly in chloroplast in wild-type N. benthamiana cells. In the presence of RSV SP, most PsbP was recruited into cytoplasm of the assayed cells. In addition, accumulation of SP during RSV infection resulted in alterations of chloroplast structure and function. Our findings shed light on the molecular mechanism underlying RSV disease symptom development.