Volume electron microscopy reconstruction uncovers a physical barrier that limits virus to phloem.

Most plant reoviruses are phloem-limited, but the mechanism has remained unknown for more than half a century. Southern rice black-streaked dwarf virus (Fijivirus, Reoviridae) causes phloem-derived tumors, where its virions, genomes, and proteins accumulate, and it was used as a model to explore how its host plant limits the virus within its phloem. High-throughput volume electron microscopy revealed that only sieve plate pores and flexible gateways rather than plasmodesmata had a sufficiently large size exclusion limit (SEL) to accommodate virions and potentially serve as pathways of virion movement. The large SEL gateways were enriched within the proliferated sieve element (SE) layers of tumors. The lack of such connections out of the SE-enriched regions of tumors defined a size-dependent physical barrier to high flux transportation of virions. A working model is proposed to demonstrate the mechanism underlying limitation of virus within phloem.

A new demethylase gene, OsDML4, is involved in high temperature-increased grain chalkiness in rice.

High temperature (HT) can affect the accumulation of seed storage materials and cause adverse effects on the yield and quality of rice. DNA methylation plays an important role in plant growth and development. Here, we identified a new demethylase gene OsDML4 and discovered its function in cytosine demethylation to affect endosperm formation. Loss of function of OsDML4 induced chalky endosperm only under HT and dramatically reduced the transcription and accumulation of glutelins and 16 kDa prolamin. The expression of two transcription factor genes RISBZ1 and RPBF was significantly decreased in the osdml4 mutants, which caused adverse effects on the formation of protein bodies (PBs) with greatly decreased PB-II number, and incomplete and abnormally shaped PB-IIs. Whole-genome bisulfite sequencing analysis of seeds at 15 d after pollination revealed much higher global methylation levels of CG, CHG, and CHH contexts in the osdml4 mutants compared with the wild type. Moreover, the RISBZ1 promoter was hypermethylated but the RPBF promoter was almost unchanged under HT. No significant difference was detected between the wild type and osdml4 mutants under normal temperature. Our study demonstrated a novel OsDML4-mediated DNA methylation involved in the formation of chalky endosperm only under HT and provided a new perspective in regulating endosperm development and the accumulation of seed storage proteins in rice.

MicroRNAs play important roles in regulating the rapid growth of the Phyllostachys edulis culm internode.

Moso bamboo (Phyllostachys edulis) is a fast-growing species with uneven growth and lignification from lower to upper segments within one internode. MicroRNAs (miRNAs) play a vital role in post-transcriptional regulation in plants. However, how miRNAs regulate fast growth in bamboo internodes is poorly understood. In this study, one moso bamboo internode was divided during early rapid growth into four segments called F4 (bottom) to F1 (upper) and these were then analysed for transcriptomes, miRNAs and degradomes. The F4 segment had a higher number of actively dividing cells as well as a higher content of auxin (IAA), cytokinin (CK) and gibberellin (GA) compared with the F1 segment. RNA-seq analysis showed DNA replication and cell division-associated genes highly expressed in F4 rather than in F1. In total, 63 miRNAs (DEMs) were identified as differentially expressed between F4 and F1. The degradome and the transcriptome indicated that many downstream transcription factors and hormonal responses genes were modulated by DEMs. Several miR-target interactions were further validated by tobacco co-infiltration. Our findings give new insights into miRNA-mediated regulatory pathways in bamboo, and will contribute to a comprehensive understanding of the molecular mechanisms governing rapid growth.

A novel pathogenicity determinant hijacks maize catalase 1 to enhance viral multiplication and infection.

Pathogens have evolved various strategies to overcome host immunity for successful infection. Maize chlorotic mottle virus (MCMV) can cause lethal necrosis in maize (Zea mays) when it coinfects with a virus in the Potyviridae family. However, the MCMV pathogenicity determinant remains largely unknown. Here we show that the P31 protein of MCMV is important for viral accumulation and essential for symptom development. Ectopic expression of P31 using foxtail mosaic virus or potato virus X induced necrosis in systemically infected maize or Nicotiana benthamiana leaves. Maize catalases (CATs) were shown to interact with P31 in yeast and in planta. P31 accumulation was elevated through its interaction with ZmCAT1. P31 attenuated the expression of salicylic acid (SA)-responsive pathogenesis-related (PR) genes by inhibiting catalase activity during MCMV infection. In addition, silencing of ZmCATs using a brome mosaic virus-based gene silencing vector facilitated MCMV RNA and coat protein accumulation. This study reveals an important role for MCMV P31 in counteracting host defence and inducing systemic chlorosis and necrosis. Our results have implications for understanding the mechanisms in defence and counter-defence during infection of plants by various pathogens.

Rice black-streaked dwarf virus-encoded P5-1 regulates the ubiquitination activity of SCF E3 ligases and inhibits jasmonate signaling to benefit its infection in rice.

SCF (Skp1/Cullin1/F-box) complexes are key regulators of many cellular processes. Viruses encode specific factors to interfere with or hijack these complexes and ensure their infection in plants. The molecular mechanisms controlling this interference/hijack are currently largely unknown. Here, we present evidence of a novel strategy used by Rice black-streaked dwarf virus (RBSDV) to regulate ubiquitination in rice (Oryza sativa) by interfering in the activity of OsCSN5A. We also show that RBSDV P5-1 specifically affects CSN-mediated deRUBylation of OsCUL1, compromising the integrity of the SCFCOI1 complex. We demonstrate that the expressions of jasmonate (JA) biosynthesis-associated genes are not inhibited, whereas the expressions of JA-responsive genes are down-regulated in transgenic P5-1 plants. More importantly, application of JA to P5-1 transgenic plants did not reduce their susceptibility to RBSDV infection. Our results suggest that P5-1 inhibits the ubiquitination activity of SCF E3 ligases through an interaction with OsCSN5A, and hinders the RUBylation/deRUBylation of CUL1, leading to an inhibition of the JA response pathway and an enhancement of RBSDV infection in rice.

Interaction between southern rice black-streaked dwarf virus minor core protein P8 and a rice zinc finger transcription factor.

The fijivirus southern rice black-streaked dwarf virus (SRBSDV) causes one of the most serious viral diseases of rice in China and Vietnam. To better understand the molecular basis of SRBSDV infection, a yeast two-hybrid screen of a rice cDNA library was carried out using P8, a minor core protein of SRBSDV, as the bait. A rice Cys2His2-type zinc finger protein (OsZFP) was found to interact with SRBSDV P8. A strong interaction between SRBSDV P8 and OsZFP was then confirmed by pull-down assays, and bimolecular fluorescence complementation assays showed that the in vivo interaction was specifically localized in the nucleus of plant cells. Using a series of deletion mutants, it was shown that both the NTP-binding region of P8 and the first two zinc fingers of OsZFP were crucial for their interaction in plant cells. The localization in the nucleus and activation of transcription in yeast supports the notion that OsZFP is a transcription factor. SRBSDV P8 may play an important role in fijiviral infection and symptom development by interfering with the host transcription activity of OsZFP.

Functional identification of two minor capsid proteins from Chinese wheat mosaic virus using its infectious full-length cDNA clones.

Full-length cDNA clones of Chinese wheat mosaic virus (CWMV) RNA1 and RNA2 were produced from single reverse transcription PCR reactions and transcripts were shown to be infectious in both wheat and Nicotiana benthamiana. An efficient and reliable agro-infiltration method was then developed for reverse genetic assays in N. benthamiana. Inoculation of infectious cDNA clones resulted in obvious chlorotic symptoms, and CWMV viral genomic RNAs, capsid protein (CP)-related proteins, and typical rod-shaped particles were detectable on the inoculated and upper leaves, similar to those of WT virus. The optimal temperature for virus multiplication was 12 °C, but the optimum for systematic infection in plants was 17 °C. Mutant clones that abolished the N- or C-terminal extensions of the major CP did not inhibit systemic infection or the formation of rod-shaped particles but sometimes modified the symptoms in inoculated plants. These results suggest that the two minor CP-related proteins of CWMV are dispensable for viral infection, replication, systemic movement and virion assembly in plants.

Proteomic dissection of plant responses to various pathogens.

During their growth and development, plants are vulnerable to the effects of a variety of pathogens. Proteomics technology plays an important role in research studies of plant defense mechanisms by mining the expression changes of proteins in response to various biotic stresses. This review article provides a comprehensive overview of the latest developments in international proteomic research on plant biotic stress. It summarizes the methods commonly used in plant proteomic research to investigate biotic stress, analyze the protein responses of plants in adverse conditions, and reviews the applications of proteomics combined with transgenic technology in plant protection.

Genome-wide identification and evolutionary analysis of positively selected miRNA genes in domesticated rice.

The next-generation sequencing of tens to hundreds of plant genotypes made the uncovering of miRNA genes evolution available at the genome-wide level. Using the combinations of population genetics and evolutionary biology approaches, we have identified 21 miRNA loci having significant negative Tajima's D and Fu and Li's D* and F* values, of which 14 miRNAs (ps-miRNAs) showing clear signatures of positive selection in domesticated rice. The average sequence diversity (π) of the 21 miRNAs in cultivated rice is only 13.8 % of that in their wild progenitors. Interestingly, protein-coding genes immediately flanking these ps-miRNAs are apparently under weaker selective constraints. Totally, the 21 miRNAs are predicted to target 68 mRNA genes, of which 12 targets are estimated to have endured positive selection during rice evolution. In addition, the expression pattern and potential biological functions of ps-miRNAs targets are further investigated by searching published micro-array data and different mutant databases, respectively. We conclude that miRNAs, like protein-coding genes, should be crucial for driving rice evolution. These analyses may deepen our understanding on the miRNA genes evolution and functions during rice domestication.

Nuclear-cytoplasmic partitioning of cucumber mosaic virus protein 2b determines the balance between its roles as a virulence determinant and an RNA-silencing suppressor.

UNLABELLED: The Cucumber Mosaic Virus (CMV) 2b protein is an RNA-silencing suppressor that plays roles in CMV accumulation and virulence. The 2b proteins of subgroup IA CMV strains partition between the nucleus and cytoplasm, but the biological significance of this is uncertain. We fused an additional nuclear localization signal (NLS) to the 2b protein of subgroup IA strain Fny-CMV to create 2b-NLS and tested its effects on subcellular distribution, silencing, and virulence. The additional NLS enhanced 2b protein nuclear and nucleolar accumulation, but nuclear and nucleolar enrichment correlated with markedly diminished silencing suppressor activity in patch assays and abolished 2b protein-mediated disruption of microRNA activity in transgenic Arabidopsis. Nucleus/nucleolus-localized 2b protein possesses at least some ability to inhibit antiviral silencing, but this was not sufficient to prevent recovery from disease in younger, developing leaves in Arabidopsis. However, enhanced nuclear and nucleolar accumulation of 2b increased virulence and accelerated symptom appearance in older leaves. Experiments with Arabidopsis lines carrying mutant Dicer-like alleles demonstrated that compromised suppressor activity explained the diminished ability of 2b-NLS to enhance virus accumulation. Remarkably, the increased virulence that 2b-NLS engendered was unrelated to effects on microRNA- or short interfering RNA-regulated host functions. Thus, although nucleus- and nucleolus-localized 2b protein is less efficient at silencing suppression than cytoplasm-localized 2b, it enhances CMV virulence. We propose that partitioning of the 2b protein between the cytoplasmic and nuclear/nucleolar compartments allows CMV to regulate the balance between virus accumulation and damage to the host, presumably to maximize the benefit for the virus. IMPORTANCE: In this work, the main finding is that nucleus/nucleolus-localized 2b protein is strongly associated with CMV virulence, which is independent of its effect on small RNA pathways. Moreover, this work supports the contention that the silencing suppressor activity of CMV 2b protein is predominantly exerted by that portion of the 2b protein residing in the cytoplasm. Thus, we propose that partitioning of the 2b protein between the cytoplasmic and nuclear/nucleolar compartments allows CMV to regulate the balance between virus accumulation and damage to the host, presumably to maximize the benefit for the virus.

Characterization of homologous and heterologous interactions between viroplasm proteins P6 and P9-1 of the fijivirus southern rice black-streaked dwarf virus.

P6 of southern rice black-streaked dwarf virus (SRBSDV) is a multifunctional protein that is involved in the formation of viroplasms by interacting with P5-1. Here, we used yeast two-hybrid and bimolecular fluorescence complementation assays to show that there were homologous and heterologous interactions between SRBSDV P6 and P9-1 in yeast and plant cells. Mutational analysis showed that the N-terminal region (residues 1-93) of P6 was necessary for the interaction between P6 and P9-1. Self-interactions only occurred between the full-length P6 or P9-1. P9-1 was able to form viroplasm-like inclusion structures alone in the absence of other viral proteins.

P5-2 of rice black-streaked dwarf virus is a non-structural protein targeted to chloroplasts.

The genome segment S5 of rice black-streaked dwarf virus (genus Fijivirus, family Reoviridae) is functionally bicistronic in infected plants. It has a conserved second ORF (P5-2) partially overlapping the major ORF in a different reading frame, but its function remains unknown. P5-2 was detected in infected plants, but not in purified viral particles by Western blotting, indicating that it is a non-structural protein. In immunoelectron microscopy, polyclonal antibodies against P5-2 specifically labelled chloroplasts of infected rice plants. When P5-2 fused with green fluorescent protein was transiently expressed in leaves of Nicotiana benthamiana, fluorescence was also co-localized with chloroplasts. Experiments with deletion mutants of P5-2 showed that its N-terminal part was responsible for its targeting to chloroplasts.

Domains of the cucumber mosaic virus 2b silencing suppressor protein affecting inhibition of salicylic acid-induced resistance and priming of salicylic acid accumulation during infection.

The cucumber mosaic virus (CMV) 2b silencing suppressor protein allows the virus to overcome resistance to replication and local movement in inoculated leaves of plants treated with salicylic acid (SA), a resistance-inducing plant hormone. In Arabidopsis thaliana plants systemically infected with CMV, the 2b protein also primes the induction of SA biosynthesis during this compatible interaction. We found that CMV infection of susceptible tobacco (Nicotiana tabacum) also induced SA accumulation. Utilization of mutant 2b proteins expressed during infection of tobacco showed that the N- and C-terminal domains, which had previously been implicated in regulation of symptom induction, were both required for subversion of SA-induced resistance, while all mutants tested except those affecting the putative phosphorylation domain had lost the ability to prime SA accumulation and expression of the SA-induced marker gene PR-1.

Tumours induced by a plant virus are derived from vascular tissue and have multiple intercellular gateways that facilitate virus movement.

Structural studies showed that tumours induced by Southern rice black-streaked dwarf virus (SRBSDV; genus Fijivirus, family Reoviridae) were highly organized, modified phloem, composed of sclerenchyma, vessels, hyperplastic phloem parenchyma and sieve elements (SEs). Only parenchyma and SEs were invaded by the virus. There was a special region that consisted exclusively of SEs without the usual companion cells and a new flexible type of intercellular gateway was observed on all SE-SE interfaces in this region. These flexible gateways significantly increased the intercellular contacts and thus enhanced potential symplastic transport in the tumour. Flexible gateways were structurally similar to compressed plasmodesmata but were able to accommodate complete SRBSDV virions (~80 nm diameter). Virions were also found in sieve-pore gateways, providing strong evidence for the movement of a virus with large virions within phloem tissue and suggesting that the unusual neovascularization of plant virus-induced tumours facilitated virus spread. A working model for the spread of tumour-inducing reoviruses in plants is presented.

Analysis of small RNAs derived from Chinese wheat mosaic virus.

The virus-derived small interfering RNAs (vsiRNAs) of Chinese wheat mosaic virus (CWMV), a member of the genus Furovirus, were characterised from wheat plants by deep sequencing. CWMV vsiRNAs of 21-22 nt in length predominated, suggesting that there might be a conserved mechanism of DCL2 and DCL4 involvement in the biogenesis of vsiRNAs, as well as a common RNA silencing pathway in CWMV-infected wheat plants. The 5'-terminal base of vsiRNAs was biased towards A/U, suggesting that CWMV vsiRNAs might be loaded into diverse AGO-containing RISCs to disturb the gene expression of host plants. Possible targets for some of the vsiRNAs were predicted.

Rice black-streaked dwarf virus genome segment S5 is a bicistronic mRNA in infected plants.

Rice black-streaked dwarf virus (RBSDV) is a recognized member of the genus Fijivirus, family Reoviridae. Genome segment S5 has a putative second ORF partially overlapping the major ORF but in a different reading frame. This putative ORF is present in a published sequence and in two Chinese isolates now sequenced. Antibodies were raised against purified P5-1 and P5-2 fusion proteins expressed in a prokaryotic system. In western blots, these antibodies reacted with proteins of about 106 and 27 kDa, respectively, as predicted by sequence analysis. In immunoelectron microscopy, antibodies to P5-1 reacted with viroplasms, indicating that P5-1 is a component of viroplasms, but no labeling was observed with P5-2 antisera. Northern blot assays showed that the genome segment S5 was transcribed as a single mRNA with no subgenomic RNA. These results show that S5 is functionally bicistronic in infected plants. Possible translational mechanisms for P5-2 are discussed.

Interactions between the P6 and P5-1 proteins of southern rice black-streaked dwarf fijivirus in yeast and plant cells.

Southern rice black-streaked dwarf virus (SRBSDV) is a recently described member of the genus Fijivirus, family Reoviridae. The roles of the proteins encoded by the SRBSDV genome have rarely been studied. In a yeast two-hybrid (YTH) assay in which SRBSDV P6, a putatively multifunctional protein, was used as bait and an SRBSDV cDNA library was used as prey, there was a strong interaction between the P6 and P5-1 proteins. The interaction was confirmed by bimolecular fluorescence complement (BiFC) assay in plant cells. YTH analysis using truncated mutants showed that the N-terminal region (amino acids 9-231) of P5-1 is necessary for binding P5-1 to P6 and that the N-terminal fragment (amino acids 1-93) of P6 is necessary for its interaction with P5-1. SRBSDV P5-1 formed granules positioned at the cell periphery in Nicotiana benthamiana leaves; P6 was present in both the cytoplasm and the nucleus and formed punctate bodies associated with the cell periphery. Immunogold labeling showed that both P6 and P5-1 localized within viroplasms in infected cells of rice plants. These results suggest that the interaction between P5-1 and P6 of SRBSDV may be involved in the formation of viroplasms.

Simultaneous Detection and Survey of Three Rice Viruses in China.

Rice black-streaked dwarf virus, Rice stripe virus, and Southern rice black-streaked dwarf virus (SRBSDV) have been epidemic in large areas of China where rice is grown, causing significant losses of rice yield in recent years. These viral diseases sometimes occur in the same regions, and even in the same fields, making it difficult to detect and diagnose the viral pathogens. A set of primers specific to the genes encoding the capsid proteins of the three viruses were designed, and a multiple one-step reverse-transcription polymerase chain reaction protocol was developed. The method proved to be simple, rapid, and sensitive. It was used to detect the viruses in samples of rice, maize, small brown planthoppers, and white-backed planthoppers collected from different regions of China, showing that it is suitable for routine diagnosis. A simultaneous survey of the three viruses was further conducted by this method throughout Zhejiang Province, Eastern China. The results showed that both RBSDV and RSV had continued to spread and that the newly emerging virus, SRBSDV, was present in at least 27 counties or cities, suggesting that more effort is needed to monitor and control the threat from these three viral diseases.

A fijiviral nonstructural protein triggers cell death in plant and bacterial cells via its transmembrane domain.

Southern rice black-streaked dwarf virus (SRBSDV; Fijivirus, Reoviridae) has become a threat to cereal production in East Asia in recent years. Our previous cytopathologic studies have suggested that SRBSDV induces a process resembling programmed cell death in infected tissues that results in distinctive growth abnormalities. The viral product responsible for the cell death, however, remains unknown. Here P9-2 protein, but not its RNA, was shown to induce cell death in Escherichia coli and plant cells when expressed either locally with a transient expression vector or systemically using a heterologous virus. Both computer prediction and fluorescent assays indicated that the viral nonstructural protein was targeted to the plasma membrane (PM) and further modification of its subcellular localization abolished its ability to induce cell death, indicating that its PM localization was required for the cell death induction. P9-2 was predicted to harbour two transmembrane helices within its central hydrophobic domain. A series of mutation assays further showed that its central transmembrane hydrophobic domain was crucial for cell death induction and that its conserved F90, Y101, and L103 amino acid residues could play synergistic roles in maintaining its ability to induce cell death. Its homologues in other fijiviruses also induced cell death in plant and bacterial cells, implying that the fijiviral nonstructural protein may trigger cell death by targeting conserved cellular factors or via a highly conserved mechanism.

Identification, Characterization, and Distribution of Southern rice black-streaked dwarf virus in Vietnam.

A novel dwarf and twisting syndrome first observed on rice in Nghe An Province, Vietnam, in 2009 has spread rapidly to the other 19 provinces of North and Central Vietnam. Infected rice plants showed stunting, darkening of leaves, twisting of leaf tips, and splitting of leaf margins. At a later stage, white waxy enations that eventually turned black were observed on the underside of leaf blades, leaf sheaths, and culms. The disease also infected maize after rice was harvested. Infected maize plants were stunted and dark green with small enations along the minor veins on the back of leaves. The disease agent has now been identified as Southern rice black-streaked dwarf virus (SRBSDV) recently reported from Southern China. Typical fijivirus viroplasms containing crystalline arrayed spherical virions approximately 70 to 75 nm in diameter were observed under the electron microscope in ultrathin sections of infected rice leaves. The virus was transmitted to rice and maize seedlings by the white-backed planthopper (Sogatella furcimera). A one-step reverse transcription-polymerase chain reaction (RT-PCR) protocol was used to confirm the presence of SRBSDV in 477 samples of rice or maize from 29 provinces among 5 agroecological regions in North and Central Vietnam. Rice black-streaked dwarf virus was not detected in these samples. Partial sequences of RNA segments 4 and 10 from several isolates showed very low genetic divergences between isolates from Vietnam and China, suggesting a common origin, and phylogenetic analysis confirmed the placement of SRBSDV as a distinct virus within subgroup 2 of the genus Fijivirus.