Zihao Xia

Characterization of Small Interfering RNAs Derived from Sugarcane Mosaic Virus in Infected Maize Plants by Deep Sequencing

RNA silencing is a conserved surveillance mechanism against viruses in plants. It is mediated by Dicer-like (DCL) proteins producing small interfering RNAs (siRNAs), which guide specific Argonaute (AGO)-containing complexes to inactivate viral genomes and may promote the silencing of host mRNAs. In this study, we obtained the profile of virus-derived siRNAs (vsiRNAs) from Sugarcane mosaic virus (SCMV) in infected maize (Zea mays L.) plants by deep sequencing. Our data showed that vsiRNAs which derived almost equally from sense and antisense SCMV RNA strands accumulated preferentially as 21- and 22-nucleotide (nt) species and had an adenosine bias at the 5′-terminus. The single-nucleotide resolution maps revealed that vsiRNAs were almost continuously but heterogeneously distributed throughout the SCMV genome and the hotspots of sense and antisense strands were mainly distributed in the HC-Pro coding region. Moreover, dozens of host transcripts targeted by vsiRNAs were predicted, several of which encode putative proteins involved in ribosome biogenesis and in biotic and abiotic stresses. We also found that ZmDCL2 mRNAs were up-regulated in SCMV-infected maize plants, which may be the cause of abundant 22-nt vsiRNAs production. However, ZmDCL4 mRNAs were down-regulated slightly regardless of the most abundant 21-nt vsiRNAs. Our results also showed that SCMV infection induced the accumulation of AGO2 mRNAs, which may indicate a role for AGO2 in antiviral defense. To our knowledge, this is the first report on vsiRNAs in maize plants.

Rapid and sensitive detection of Banana bunchy top virus by loop-mediated isothermal amplification

A sensitive loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of Banana bunchy top virus (BBTV) infection. The reaction was performed in a single tube at 63°C for 90 min, with an improved closed-tube detection system by adding the SYBR Green I dye to the inside of the tube lid prior to amplification. The detection limit of the LAMP assay was approximately 1 pg/μl plasmid DNA when mixed with extracted DNA from healthy banana plant, and no cross-reaction with other banana-infected pathogens was observed. Real-time turbidimetry was used to monitor the amplification result in the tubes, and it was shown that this LAMP assay was about 100-fold more sensitive than PCR. The results demonstrated that this LAMP method should be useful for both banana disease monitoring and mass propagation of virus-free banana plantlets.

Rapid and Efficient Isolation of High-Quality Small RNAs from Recalcitrant Plant Species Rich in Polyphenols and Polysaccharides

Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are important regulators of plant development and gene expression. The acquisition of high-quality small RNAs is the first step in the study of its expression and function analysis, yet the extraction method of small RNAs in recalcitrant plant tissues with various secondary metabolites is not well established, especially for tropical and subtropical plant species rich in polysaccharides and polyphenols. Here, we developed a simple and efficient method for high quality small RNAs extraction from recalcitrant plant species. Prior to RNA isolation, a precursory step with a CTAB-PVPP buffer system could efficiently remove compounds and secondary metabolites interfering with RNAs from homogenized lysates. Then, total RNAs were extracted by Trizol reagents followed by a differential precipitation of high-molecular-weight (HMW) RNAs using polyethylene glycol (PEG) 8000. Finally, small RNAs could be easily recovered from supernatant by ethanol precipitation without extra elimination steps. The isolated small RNAs from papaya showed high quality through a clear background on gel and a distinct northern blotting signal with miR159a probe, compared with other published protocols. Additionally, the small RNAs extracted from papaya were successfully used for validation of both predicted miRNAs and the putative conserved tasiARFs. Furthermore, the extraction method described here was also tested with several other subtropical and tropical plant tissues. The purity of the isolated small RNAs was sufficient for such applications as end-point stem-loop RT-PCR and northern blotting analysis, respectively. The simple and feasible extraction method reported here is expected to have excellent potential for isolation of small RNAs from recalcitrant plant tissues rich in polyphenols and polysaccharides.

Synergistic infection of two viruses MCMV and SCMV increases the accumulations of both MCMV and MCMV-derived siRNAs in maize

The co-infection of Maize chlorotic mottle virus (MCMV) and Sugarcane mosaic virus (SCMV) can cause maize lethal necrosis. However, the mechanism underlying the synergistic interaction between these two viruses remains elusive. In this study, we found that the co-infection of MCMV and SCMV increased the accumulation of MCMV. Moreover, the profiles of virus-derived siRNAs (vsiRNAs) from MCMV and SCMV in single- and co-infected maize plants were obtained by high-throughput sequencing. Our data showed that synergistic infection of MCMV and SCMV increased remarkably the accumulation of vsiRNAs from MCMV, which were mainly 22 and 21 nucleotides in length. The single-nucleotide resolution maps of vsiRNAs revealed that vsiRNAs were almost continuously but heterogeneously distributed throughout MCMV and SCMV genomic RNAs, respectively. Moreover, we predicted and annotated dozens of host transcript genes targeted by vsiRNAs. Our results also showed that maize DCLs and several AGOs RNAs were differentially accumulated in maize plants with different treatments (mock, single or double inoculations), which were associated with the accumulation of vsiRNAs. Our findings suggested possible roles of vsiRNAs in the synergistic interaction of MCMV and SCMV in maize plants.

One-step reverse transcription loop-mediated isothermal amplification for the detection of Maize chlorotic mottle virus in maize.

Maize chlorotic mottle virus (MCMV) is spreading in many regions worldwide, causing maize lethal necrosis when co-infected with a potyvirid. In this study, one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect MCMV in maize. A set of four specific primers was designed based on the conserved coat protein gene sequences of MCMV. The RT-LAMP could be completed within 60min under isothermal condition at 63°C. The sensitivity test showed that the RT-LAMP was about 10-fold more sensitive than RT-PCR and no cross-reactivity was detected with other viral pathogens infecting maize in China. Moreover, the results of RT-LAMP could be visually inspected by SYBR Green I staining in a closed-tube, facilitating high-throughput application of MCMV detection. This method was further verified by testing field-collected samples. These results suggested that the developed MCMV RT-LAMP technique is a rapid, efficient and sensitive method which could be used as a routine screen for MCMV infection.

A Violaxanthin Deepoxidase Interacts with a Viral Suppressor of RNA Silencing to Inhibit Virus Amplification

Maize violaxanthin deepoxidase interacts with the HC-Pro protein of Sugarcane mosaic virus to attenuate its RNA-silencing suppression activity, contributing to decreased viral accumulation. RNA silencing plays a critical role against viral infection. To counteract this antiviral silencing, viruses have evolved various RNA silencing suppressors. Meanwhile, plants have evolved counter-counter defense strategies against RNA silencing suppression (RSS). In this study, the violaxanthin deepoxidase protein of maize (Zea mays), ZmVDE, was shown to interact specifically with the helper component-proteinase (HC-Pro; a viral RNA silencing suppressor) of Sugarcane mosaic virus (SCMV) via its mature protein region by yeast two-hybrid assay, which was confirmed by coimmunoprecipitation in Nicotiana benthamiana cells. It was demonstrated that amino acids 101 to 460 in HC-Pro and the amino acid glutamine-292 in ZmVDE mature protein were essential for this interaction. The mRNA levels of ZmVDE were down-regulated 75% to 65% at an early stage of SCMV infection. Interestingly, ZmVDE, which normally localized in the chloroplasts and cytoplasm, could relocalize to HC-Pro-containing aggregate bodies in the presence of HC-Pro alone or SCMV infection. In addition, ZmVDE could attenuate the RSS activity of HC-Pro in a specific protein interaction-dependent manner. Subsequently, transient silencing of the ZmVDE gene facilitated SCMV RNA and coat protein accumulation. Taken together, our results suggest that ZmVDE interacts with SCMV HC-Pro and attenuates its RSS activity, contributing to decreased SCMV accumulation. This study demonstrates that a host factor can be involved in secondary defense responses against viral infection in monocot plants.

Identification of differentially regulated maize proteins conditioning Sugarcane mosaic virus systemic infection

Sugarcane mosaic virus (SCMV) is the most important cause of maize dwarf mosaic disease. To identify maize genes responsive to SCMV infection and that may be involved in pathogenesis, a comparative proteomic analysis was performed using the first and second systemically infected leaves (termed 1 SL and 2 SL, respectively). Seventy-one differentially expressed proteins were identified in 1 SL and 2 SL upon SCMV infection. Among them, eight proteins showed the same changing patterns in both 1 SL and 2 SL. Functional annotations of regulated proteins and measurement of photosynthetic activity revealed that photosynthesis was more inhibited and defensive gene expression more pronounced in 1 SL than in 2 SL. Knockdown of regulated proteins in both 1 SL and 2 SL by a brome mosaic virus-based gene silencing vector in maize indicated that protein disulfide isomerase-like and phosphoglycerate kinase were required for optimal SCMV replication. By contrast, knockdown of polyamine oxidase (ZmPAO) significantly increased SCMV accumulation, implying that ZmPAO activity might contribute to resistance or tolerance. The results suggest that combining comparative proteomic analyses of different tissues and virus-induced gene silencing is an efficient way to identify host proteins supporting virus replication or enhancing resistance to virus infection.

Characterization of maize translational responses to sugarcane mosaic virus infection

Sugarcane mosaic virus (SCMV) frequently causes dramatic losses in maize production as the main pathogen of maize dwarf mosaic disease. It is important to understand the translational responses in maize to SCMV infection since viruses have to recruit host translation apparatus to express their proteins. However, due to technical limitations, research on virus translation lags far behind that on transcription. Here, we studied the relationship between systemic symptom expression and virus accumulation and found that both SCMV RNA and proteins accumulated rapidly during the systemic infection process in which varying degrees of chlorosis to mosaic symptoms developed on non-inoculated leaves. In addition, we applied ribosome profiling, which couples polysomal mRNA isolation with high-throughput sequencing, on the symptomatic leaves infected with SCMV to unravel the translational responses of maize to viral infection on a genome-wide scale. The results showed that only the genomic positive-stranded RNA of SCMV was involved in translation, and SCMV only occupied a small amount of translational resources of host plant at the early stage of infection. Further analyses on a global gene expression and gene ontology (GO) enrichment revealed that photosynthesis and metabolism were dramatically repressed at both transcriptional and translational levels. Altogether, our results laid a foundation for dissecting the molecular mechanism of plant translational responses to viral infection.

Identification of miRNAs and their targets in maize in response to Sugarcane mosaic virus infection

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that play essential regulatory roles in plant development and environmental stress responses. Maize (Zea mays L.) is a global economically important food and forage crop. To date, a number of maize miRNAs have been identified as being involved in plant development and stress responses. However, the miRNA-mediated gene regulatory networks responsive to virus infections in maize remain largely unknown. In this study, the profiles of small RNAs in buffer- and Sugarcane mosaic virus (SCMV)-inoculated maize plants were obtained by high-throughput sequencing, respectively. A total of 154 known miRNAs and 213 novel miRNAs were profiled and most of the miRNAs identified were differentially expressed after SCMV infection. In addition, 70 targets of 13 known miRNAs and 3 targets of a novel miRNA were identified by degradome analysis. The results of Northern blotting and quantitative real-time PCR showed that the expression levels of the selected miRNAs and their targets were mostly influenced by SCMV infection at 12 days post inoculation, including up-regulation of miR168 and miR528, and down-regulation of miR159, miR397 and miR827. These results provide new insights into the regulatory networks of miRNAs and their targets in maize plants responsive to SCMV infection.