Integrated ATAC-seq and RNA-seq data analysis identifies transcription factors related to rice stripe virus infection in Oryza sativa.

Animal studies have shown that virus infection causes changes in host chromatin accessibility, but little is known about changes in chromatin accessibility of plants infected by viruses and its potential impact. Here, rice infected by rice stripe virus (RSV) was used to investigate virus-induced changes in chromatin accessibility. Our analysis identified a total of 6462 open- and 3587 closed-differentially accessible chromatin regions (DACRs) in rice under RSV infection by ATAC-seq. Additionally, by integrating ATAC-seq and RNA-seq, 349 up-regulated genes in open-DACRs and 126 down-regulated genes in closed-DACRs were identified, of which 34 transcription factors (TFs) were further identified by search of upstream motifs. Transcription levels of eight of these TFs were validated by reverse transcription-PCR. Importantly, four of these TFs (OsWRKY77, OsWRKY28, OsZFP12 and OsERF91) interacted with RSV proteins and are therefore predicted to play important roles in RSV infection. This is the first application of ATAC-seq and RNA-seq techniques to analyse changes in rice chromatin accessibility caused by RSV infection. Integrating ATAC-seq and RNA-seq provides a new approach to select candidate TFs in response to virus infection.

Turnip mosaic virus NIb weakens the function of eukaryotic translation initiation factor 6 facilitating viral infection in Nicotiana benthamiana.

Viruses rely completely on host translational machinery to produce the proteins encoded by their genes. Controlling translation initiation is important for gaining translational advantage in conflicts between the host and virus. The eukaryotic translation initiation factor 4E (eIF4E) has been reported to be hijacked by potyviruses for virus multiplication. The role of translation regulation in defence and anti-defence between plants and viruses is not well understood. We report that the transcript level of eIF6 was markedly increased in turnip mosaic virus (TuMV)-infected Nicotiana benthamiana. TuMV infection was impaired by overexpression of N. benthamiana eIF6 (NbeIF6) either transiently expressed in leaves or stably expressed in transgenic plants. Polysome profile assays showed that overexpression of NbeIF6 caused the accumulation of 40S and 60S ribosomal subunits, the reduction of polysomes, and also compromised TuMV UTR-mediated translation, indicating a defence role for upregulated NbeIF6 during TuMV infection. However, the polysome profile in TuMV-infected leaves was not identical to that in leaves overexpressing NbeIF6. Further analysis showed that TuMV NIb protein, the RNA-dependent RNA polymerase, interacted with NbeIF6 and interfered with its effect on the ribosomal subunits, suggesting that NIb might have a counterdefence role. The results propose a possible regulatory mechanism at the translation level during plant-virus interaction.

Pepper mild mottle virus coat protein interacts with pepper chloroplast outer envelope membrane protein OMP24 to inhibit antiviral immunity in plants.

Pepper mild mottle virus (PMMoV) is a devastating viral pathogen of pepper (Capsicum annuum) but it is unclear whether and how peppers protect against PMMoV infection. The expression of the chloroplast outer membrane protein 24 (OMP24) of C. annuum was upregulated under PMMoV infection and it interacted with PMMoV coat protein (CP). Silencing of OMP24 in either C. annuum or Nicotiana benthamiana facilitated PMMoV infection, whereas overexpression of N. benthamiana OMP24 in transgenic plants inhibited PMMoV infection. Both C. annuum OMP24 (CaOMP24) and N. benthamiana OMP24 (NbOMP24) localized to the chloroplast and have a moderately hydrophobic transmembrane domain that is necessary for their localization. Overexpression of CaOMP24 induced stromules, perinuclear chloroplast clustering, and accumulation of reactive oxygen species (ROS), the typical defense responses of chloroplasts transferring the retrograde signaling to the nucleus to regulate resistance genes. The expression of PR1 and PR2 was also upregulated significantly in plants overexpressing OMP24. Self-interaction of OMP24 was demonstrated and was required for OMP24-mediated plant defense. Interaction with PMMoV CP interfered with the self-interaction of OMP24 and impaired OMP24-induced stromules, perinuclear chloroplast clustering and ROS accumulation. The results demonstrate the defense function of OMP24 in pepper during viral infection and suggest a possible mechanism by which PMMoV CP modulates the plant defense to facilitate viral infection.

A Novel miRNA in Rice Associated with the Low Seed Setting Rate Symptom of Rice Stripe Virus.

MicroRNAs play key regulatory roles in plant development. The changed pattern of miRNA expression is involved in the production of viral symptoms. Here, we showed that a small RNA, Seq119, a putative novel microRNA, is associated with the low seed setting rate, a viral symptom of rice stripe virus (RSV)-infected rice. The expression of Seq 119 was downregulated in RSV-infected rice. The overexpression of Seq119 in transgenic rice plants did not cause any obvious phenotypic changes in plant development. When the expression of Seq119 was suppressed in rice plants either by expressing a mimic target or by CRISPR/Cas editing, seed setting rates were extremely low, similar to the effects of RSV infection. The putative targets of Seq119 were then predicted. The overexpression of the target of Seq119 in rice caused a low seed setting rate, similar to that in Seq119-suppressed or edited rice plants. Consistently, the expression of the target was upregulated in Seq119-suppressed and edited rice plants. These results suggest that downregulated Seq119 is associated with the low seed setting rate symptom of the RSV in rice.

Downregulation of Light-Harvesting Complex II Induces ROS-Mediated Defense Against Turnip Mosaic Virus Infection in Nicotiana benthamiana.

The light-harvesting chlorophyll a/b complex protein 3 (LHCB3) of photosystem II plays important roles distributing the excitation energy and modulating the rate of state transition and stomatal response to abscisic acid. However, the functions of LHCB3 in plant immunity have not been well investigated. Here, we show that the expression of LHCB3 in Nicotiana benthamiana (NbLHCB3) was down-regulated by turnip mosaic virus (TuMV) infection. When NbLHCB3 was silenced by tobacco rattle virus-induced gene silencing, systemic infection of TuMV was inhibited. H2O2 was over-accumulated in NbLHCB3-silenced plants. Chemical treatment to inhibit or eliminate reactive oxygen species (ROS) impaired the resistance of the NbLHCB3-silenced plants to TuMV infection. Co-silencing of NbLHCB3 with genes involved in ROS production compromised the resistance of plants to TuMV but co-silencing of NbLHCB3 with genes in the ROS scavenging pathway increased resistance to the virus. Transgenic plants overexpressing NbLHCB3 were more susceptible to TuMV. These results indicate that downregulation of NbLHCB3 is involved in defense against TuMV by inducing ROS production.

Turnip mosaic virus impairs perinuclear chloroplast clustering to facilitate viral infection.

Chloroplasts play crucial roles in plant defence against viral infection. We now report that chloroplast NADH dehydrogenase-like (NDH) complex M subunit gene (NdhM) was first up-regulated and then down-regulated in turnip mosaic virus (TuMV)-infected N. benthamiana. NbNdhM-silenced plants were more susceptible to TuMV, whereas overexpression of NbNdhM inhibited TuMV accumulation. Overexpression of NbNdhM significantly induced the clustering of chloroplasts around the nuclei and disturbing this clustering facilitated TuMV infection, suggesting that the clustering mediated by NbNdhM is a defence against TuMV. It was then shown that NbNdhM interacted with TuMV VPg, and that the NdhMs of different plant species interacted with the proteins of different viruses, implying that NdhM may be a common target of viruses. In the presence of TuMV VPg, NbNdhM, which is normally localized in the nucleus, chloroplasts, cell periphery and chloroplast stromules, colocalized with VPg at the nucleus and nucleolus, with significantly increased nuclear accumulation, while NbNdhM-mediated chloroplast clustering was significantly impaired. This study therefore indicates that NbNdhM has a defensive role in TuMV infection probably by inducing the perinuclear clustering of chloroplasts, and that the localization of NbNdhM is altered by its interaction with TuMV VPg in a way that promotes virus infection.

The hypersensitive induced reaction 3 (HIR3) gene contributes to plant basal resistance via an EDS1 and salicylic acid-dependent pathway.

The hypersensitive-induced reaction (HIR) gene family is associated with the hypersensitive response (HR) that is a part of the plant defense system against bacterial and fungal pathogens. The involvement of HIR genes in response to viral pathogens has not yet been studied. We now report that the HIR3 genes of Nicotiana benthamiana and Oryza sativa (rice) were upregulated following rice stripe virus (RSV) infection. Silencing of HIR3s in N. benthamiana resulted in an increased accumulation of RSV RNAs, whereas overexpression of HIR3s in N. benthamiana or rice reduced the expression of RSV RNAs and decreased symptom severity, while also conferring resistance to Turnip mosaic virus, Potato virus X, and the bacterial pathogens Pseudomonas syringae and Xanthomonas oryzae. Silencing of HIR3 genes in N. benthamiana reduced the content of salicylic acid (SA) and was accompanied by the downregulated expression of genes in the SA pathway. Transient expression of the two HIR3 gene homologs from N. benthamiana or the rice HIR3 gene in N. benthamiana leaves caused cell death and an accumulation of SA, but did not do so in EDS1-silenced plants or in plants expressing NahG. The results indicate that HIR3 contributes to plant basal resistance via an EDS1- and SA-dependent pathway.

Sugarcane Elongin C is involved in infection by sugarcane mosaic disease pathogens.

Sugarcane (Saccharum sp. hybrid) provides the main source of sugar for humans. Sugarcane mosaic disease (SMD) is a major threat to sugarcane production. Currently, control of SMD is mainly dependent on breeding resistant cultivars through hybridization, which is time-consuming. Understanding the mechanism of viral infection may facilitate novel strategies to breed cultivars resistant to SMD and to control the disease. In this study, a wide interaction was detected between the viral VPg protein and host proteins. Several genes were screened from sugarcane cDNA library that could interact with Sugarcane streak mosaic virus VPg, including SceIF4E1 and ScELC. ScELC was predicted to be a cytoplasmic protein, but subcellular localization analysis showed it was distributed both in cytoplasmic and nuclear, and interactions were also detected between ScELC and VPg of SCMV or SrMV that reveal ScELC was widely used in the SMD pathogen infection process. ScELC and VPgs interacted in the nucleus, and may function to enhance the viral transcription rate. ScELC also interacted with SceIF4E2 both in the cytoplasm and nucleus, but not with SceIF4E1 and SceIF4E3. These results suggest that ScELC may be essential for the function of SceIF4E2, an isomer of eIF4E.