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.

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.

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.

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.

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.

Characterization of a novel annexin gene from cotton (Gossypium hirsutum cv CRI 35) and antioxidative role of its recombinant protein.

Plant annexins represent a multigene family involved in cellular elongation and development. A cDNA encoding a novel annexin was isolated from a cotton (Gossypium hirsutum) fiber cDNA library and designated GhAnx1. This gene encodes a 316 amino acid protein with a theoretical molecular mass of 36.06 kDa and a theoretical pI of 6.19. At the amino acid level, it shares high sequence similarity and has evolutionary relationships with annexins from higher plants. The purified recombinant protein expressed in Escherichia coli was used to investigate its physicochemical properties. Circular dichroism spectrum analyses showed a positive peak rising to the maximum at 196 nm and a broad negative band rounding 215 nm, suggesting that the GhAnx1 protein was prominently α-helical. The fluorescence measurements indicated that it could bind to Ca(2+) in vitro. These results demonstrated that GhAnx1 was a typical annexin protein in cotton. A bioassay experiment was conducted to analyze its potential function and showed that E. coli cells expressing GhAnx1 were protected from tert-butyl hydroperoxide (tBH) stress, suggesting that it had a potential antioxidative role. Northern blot analyses revealed that GhAnx1 was highly expressed in fibers, especially during the elongation stage, suggesting that it might be important for fiber elongation.

Improved quantification of immune-gold labeling and its use to compare the distribution of cellular factors among sub-chloroplast compartments.

Quantification of immuno-gold labeling can provide valuable information on the quantity and localization of a target within a region of interest (ROI). Background subtraction usually requires preparation of material with a deliberately reduced amount of target component often by gene knockout/knockdown. This paper reports a modified method without the need for gene knockout/knockdown, by using a region outside the ROI as a background and non-immune serum to verify the reliability of the data. An optimized parameter for use in image processing was also developed to improve semi-automatic segmentation of gold particles, by using the standard deviation of pixel intensity together with default parameters (size and intensity) to improve specificity. The modified methods were used to quantify the gold labeling of various components within chloroplasts and their 3 sub-organelle compartments (thylakoid, stroma and starch). Rubisco, actin, myosin, ß-tubulin, Endoplasmic reticulum-retention signal HDEL, Sterol methyltransferase 1, and double stranded RNA were all effectively and consistently quantified at the level of the different sub-chloroplast compartments. The approach should be applicable more widely for high resolution labelling of samples in which a background requiring gene knockout/knockdown is not a realistic option.

A class I cytosolic HSP20 of rice enhances heat and salt tolerance in different organisms.

Small heat shock proteins (sHSPs) have been thought to function as chaperones, protecting their targets from denaturation and aggregation when organisms are subjected to various biotic and abiotic stresses. We previously reported an sHSP from Oryza sativa (OsHSP20) that homodimerizes and forms granules within the cytoplasm but its function was unclear. We now show that OsHSP20 transcripts were significantly up-regulated by heat shock and high salinity but not by drought. A recombinant protein was purified and shown to inhibit the thermal aggregation of the mitochondrial malate dehydrogenase (MDH) enzyme in vitro, and this molecular chaperone activity suggested that OsHSP20 might be involved in stress resistance. Heterologous expression of OsHSP20 in Escherichia coli or Pichia pastoris cells enhanced heat and salt stress tolerance when compared with the control cultures. Transgenic rice plants constitutively overexpressing OsHSP20 and exposed to heat and salt treatments had longer roots and higher germination rates than those of control plants. A series of assays using its truncated mutants showed that its N-terminal arm plus the ACD domain was crucial for its homodimerization, molecular chaperone activity in vitro, and stress tolerance in vivo. The results supported the viewpoint that OsHSP20 could confer heat and salt tolerance by its molecular chaperone activity in different organisms and also provided a more thorough characterization of HSP20-mediated stress tolerance in O. sativa.

A black-streaked dwarf disease on rice in China is caused by a novel fijivirus.

An isolate of a plant reovirus causing severe stunting and dark leaf symptoms on rice from Guangdong, China, was similar in virion morphology and serologically related to rice black-streaked dwarf virus (RBSDV). The electrophoretic profiles of genome segments of the two viruses in agarose or polyacrylamide gel were indistinguishable. The four genome segments of the new isolate corresponding to RBSDV S7-S10 were amplified by ligation RT-PCR and sequenced. The size and organization of each genome segment was very similar to its counterparts in RBSDV, maize rough dwarf virus (MRDV), and mal de Rio Cuarto virus (MRCV). Sequence identity was greatest to RBSDV and MRDV (ranging from about 60-85% depending on the protein), but identities were always much lower than those between RBSDV and MRDV. These comparisons and phylogenetic analyses suggested that the virus represents a new species in genus Fijivirus group 2, tentatively named Rice black-streaked dwarf virus-2.