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 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.

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.

Binding between elongation factor 1A and the 3'-UTR of Chinese wheat mosaic virus is crucial for virus infection.

The Chinese wheat mosaic virus (CWMV) genome consists of two positive-strand RNAs that are required for CWMV replication and translation. The eukaryotic translation elongation factor (eEF1A) is crucial for the elongation of protein translation in eukaryotes. Here, we show that silencing eEF1A expression in Nicotiana benthamiana plants by performing virus-induced gene silencing can greatly reduce the accumulation of CWMV genomic RNAs, whereas overexpression of eEF1A in plants increases the accumulation of CWMV genomic RNAs. In vivo and in vitro assays showed that eEF1A does not interact with CWMV RNA-dependent RNA polymerase. Electrophoretic mobility shift assays revealed that eEF1A can specifically bind to the 3'-untranslated region (UTR) of CWMV genomic RNAs. By performing mutational analyses, we determined that the conserved region in the 3'-UTR of CWMV genomic RNAs is necessary for CWMV replication and translation, and that the sixth stem-loop (SL-6) in the 3'-UTR of CWMV genomic RNAs plays a key role in CWMV infection. We conclude that eEF1A is an essential host factor for CWMV infection. This finding should help us to develop new strategies for managing CWMV infections in host plants.

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.

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 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.

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.

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.

Endoplasmic reticulum remodeling induced by Wheat yellow mosaic virus infection studied by transmission electron microscopy.

Plant virus was a kind of organism lived depending on infecting viable host cell and propagated their posterity by replicating its hereditary nucleotide, transcripting into protein, assembling protein and nucleotide into virion (Ortín and Parra, 2006; Sanfaçon, 2005). Viral infection usually induces remodeling of host cell, especially endoplasmic reticulum (ER) for generating membrane packed viral factory. During the infection of Bymovirus, a kind of membranous body (MB) was generated in host cells, which is thought as an ER aggregate. In present study we performed a study on Wheat yellow mosaic virus (WYMV) induced MB by several transmission electron microscopy (TEM) based methods, including cytological observation, component analysis by immuno-gold labeling and structural analysis by electron tomography (ET). WYMV infection induced at least two morphologies of MB, including the lamella dominated morphology (lamella-MB) looked like sprawling cirrus, and the tubule dominated morphology (tubule-MB) looked like latticed network. MB was verified composing of ER as revealed by immuno-gold labeling by antibody against endoplasmic reticulum (ER) retention signal as well as by detailed observation of MB construction modules as double layer membrane. By immuno-gold labeling, both two MB morphologies (lamella-MB and tubule-MB) had same components in viral derived protein and membrane origination (from ER). Structural analysis by ET reconstruction revealed the organization of ER in MB. Lamella-MB was composed of cesER like structures arranged irregularly whereas tubule-MB was composed of tubER like structures arranged regularly. This study provided insights into the structural details in how Bymovirus utilizing host membrane system.

Chinese Wheat Mosaic Virus-Induced Gene Silencing in Monocots and Dicots at Low Temperature.

Virus-induced gene silencing (VIGS) is an important tool for functional genomics studies in plants. With this method, it is possible to target most endogenous genes and downregulate the messenger RNA (mRNA) in a sequence-specific manner. Chinese wheat mosaic virus (CWMV) has a bipartite, single-strand positive RNA genome, and can infect both wheat and Nicotiana benthamiana, and the optimal temperature for systemic infection in plants is 17°C. To assess the potential of the virus as a vector for gene silencing at low temperature, a fragment of the N. benthamiana or wheat phytoene desaturase (PDS) gene was expressed from a modified CWMV RNA2 clone and the resulting photo bleaching in infected plants was used as a reporter for silencing. Downregulation of PDS mRNA was also measured by quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR). In experiments using fragments of PDS ranging from 500 to 1500 nucleotides, insert length influenced the stability and the efficiency of VIGS. The CWMV induced silencing system was also used to suppress miR165/166 and miR3134a through expression of miRNA target mimics. The relative expression levels of mature miR165/166 and miR3134a decreased whereas the transcript levels of their target genes increased. Interestingly, we also found the CWMV-induced silencing system was more efficient compare with the vector based on Barley stripe mosaic virus (BSMV) or Foxtail mosaic virus (FoMV) in wheat or the vector based on TRV in N. benthamiana at 17°C. In summary, the CWMV vector is effective in silencing endogenous genes and miRNAs at 17°C, thereby providing a powerful tool for gene function analysis in both N. benthamiana and wheat at low temperature.

Phloem-limited reoviruses universally induce sieve element hyperplasia and more flexible gateways, providing more channels for their movement in plants.

Virion distribution and ultrastructural changes induced by the infection of maize or rice with four different reoviruses were examined. Rice black streaked dwarf virus (RBSDV, genus Fijivirus), Rice ragged stunt virus (RRSV, genus Oryzavirus), and Rice gall dwarf virus (RGDV, genus Phytoreovirus) were all phloem-limited and caused cellular hyperplasia in the phloem resulting in tumors or vein swelling and modifying the cellular arrangement of sieve elements (SEs). In contrast, virions of Rice dwarf virus (RDV, genus Phytoreovirus) were observed in both phloem and mesophyll and the virus did not cause hyperplasia of SEs. The three phloem-limited reoviruses (but not RDV) all induced more flexible gateways at the SE-SE interfaces, especially the non-sieve plate interfaces. These flexible gateways were also observed for the first time at the cellular interfaces between SE and phloem parenchyma (PP). In plants infected with any of the reoviruses, virus-like particles could be seen within the flexible gateways, suggesting that these gateways may serve as channels for the movement of plant reoviruses with their large virions between SEs or between SEs and PP. SE hyperplasia and the increase in flexible gateways may be a universal strategy for the movement of phloem-limited reoviruses.

A furoviral replicase recruits host HSP70 to membranes for viral RNA replication.

Many host factors have been identified to be involved in viral infection. However, although furoviruses cause important diseases of cereals worldwide, no host factors have yet been identified that interact with furoviral genes or participate in the viral infection cycle. In this study, both TaHSP70 and NbHSP70 were up-regulated in Chinese wheat mosaic furovirus (CWMV)-infected plants. Their overexpression and inhibition were correlated with the accumulation of viral genomic RNAs, suggesting that the HSP70 genes could be necessary for CWMV infection. The subcellular distributions of TaHSP70 and NbHSP70 were significantly affected by CWMV infection or by infiltration of RNA1 alone. Further assays showed that the viral replicase encoded by CWMV RNA1 interacts with both TaHSP70 and NbHSP70 in vivo and vitro and that its region aa167-333 was responsible for the interaction. Subcellular assays showed that the viral replicase could recruit both TaHSP70 and NbHSP70 from the cytoplasm or nucleus to the granular aggregations or inclusion-like structures on the intracellular membrane system, suggesting that both HSP70s may be recruited into the viral replication complex (VRC) to promote furoviral replication. This is the first host factor identified to be involved in furoviral infection, which extends the list and functional scope of HSP70 chaperones.

Structure and components of the globular and filamentous viroplasms induced by Rice black-streaked dwarf virus.

Viroplasms of members of the family Reoviridae are considered to be viral factories for genome replication and virion assembly. Globular and filamentous phenotypes have different components and probably have different functions. We used transmission electron microscopy and electron tomography to examine the structure and components of the two viroplasm phenotypes induced by Rice black-streaked dwarf virus (RBSDV). Immuno-gold labeling was used to localize each of the 13 RBSDV encoded proteins as well as double-stranded RNA, host cytoskeleton actin-11 and α-tubulin. Ten of the RBSDV proteins were localized in one or both types of viroplasm. P5-1, P6 and P9-1 were localized on both viroplasm phenotypes but P5-1 was preferentially associated with filaments and P9-1 with the matrix. Structural analysis by electron tomography showed that osmiophilic granules 6-8nm in diameter served as the fundamental unit for constructing both of the viroplasm phenotypes but were more densely packed in the filamentous phenotype.

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.

Rice Black-streaked Dwarf Virus Preparation and Infection on Rice.

Rice black-streaked dwarf virus (RBSDV), a member of genus Fijivirus in the family Reoviridae, infects rice, maize, barley and wheat, and can seriously affect crop yields. RBSDV is transmitted by the small brown planthopper (Laodelphax striatellus, SBPH) in a persistent manner. RBSDV has 10 linear dsRNA genomic segments, making it difficult to construct infectious clones for functional studies in plants. Here we describe a method for inoculating and maintaining RBSDV on rice in a greenhouse for use in laboratory research. The protocol uses SBPHs mass reared in the laboratory. We also describe in detail the propagation of a healthy planthopper population, the preparation of plant material, RBSDV inoculation and the evaluation of the rice after inoculation.

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.

Complete Genomic Sequence of Maize Rough Dwarf Virus, a Fijivirus Transmitted by the Small Brown Planthopper.

The nucleotide sequences of the 10 genomic segments of an Italian isolate of maize rough dwarf virus (MRDV) were determined. This first complete genomic sequence of MRDV will help understand the phylogenetic relationships among group 2 fijiviruses and especially the closely related rice black-streaked dwarf virus, which is also found to naturally infect maize.

Integrative Analysis of the microRNAome and Transcriptome Illuminates the Response of Susceptible Rice Plants to Rice Stripe Virus.

Rice stripe virus (RSV) is one of the most serious rice viruses in East Asia. To investigate how rice responds to RSV infection, we integrated miRNA expression with parallel mRNA transcription profiling by deep sequencing. A total of 570 miRNAs were identified of which 69 miRNAs (56 up-regulated and 13 down-regulated) were significantly modified by RSV infection. Digital gene expression (DGE) analysis showed that 1274 mRNAs (431 up-regulated and 843 down-regulated genes) were differentially expressed as a result of RSV infection. The differential expression of selected miRNAs and mRNAs was confirmed by qRT-PCR. Gene ontology (GO) and pathway enrichment analysis showed that a complex set of miRNA and mRNA networks were selectively regulated by RSV infection. In particular, 63 differentially expressed miRNAs were found to be significantly and negatively correlated with 160 target mRNAs. Interestingly, 22 up-regulated miRNAs were negatively correlated with 24 down-regulated mRNAs encoding disease resistance-related proteins, indicating that the host defense responses were selectively suppressed by RSV infection. The suppression of both osa-miR1423-5p- and osa-miR1870-5p-mediated resistance pathways was further confirmed by qRT-PCR. Chloroplast functions were also targeted by RSV, especially the zeaxanthin cycle, which would affect the stability of thylakoid membranes and the biosynthesis of ABA. All these modifications may contribute to viral symptom development and provide new insights into the pathogenicity mechanisms of RSV.