The TATA-box binding protein-associated factor TAF12b facilitates the degradation of type-B response regulators to negatively regulate cytokinin signaling.

Cytokinins (CKs) are one of important classes of plant hormones essential for plant growth and development. The TATA-box binding protein-associated factor 12b (TAF12b) is involved in cytokinin (CK) signaling, but its molecular and biochemical mechanisms remain unclear. In this study, TAF12b of Nicotiana benthamiana (NbTAF12b) was found to mediate CK response by directly interacting with type-B response regulators (B-RRs), which are positive regulators of CK signaling, and inhibiting their transcriptional activities. The co-factor specifically facilitated the proteasomal degradation of non-phosphorylated B-RRs by recruiting the KMD family of F-box proteins. Such interactions between TAF12b and B-RRs also occur in other plant species. Genetic transformation experiments further showed that overexpression of NbTAF12b attenuates the CK-hypersensitive phenotype conferred by NbRR1 overexpression. Taken together, these results suggest a conserved mechanism that TAF12b negatively regulates CK responses through promoting 26S proteasome-mediated degradation of B-RRs degradation in multiple plant species, which provides novel insights into the regulatory network of CK signaling in plants.

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.

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.

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.

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.

Interaction of HSP20 with a viral RdRp changes its sub-cellular localization and distribution pattern in plants.

Small heat shock proteins (sHSPs) perform a fundamental role in protecting cells against a wide array of stresses but their biological function during viral infection remains unknown. Rice stripe virus (RSV) causes a severe disease of rice in Eastern Asia. OsHSP20 and its homologue (NbHSP20) were used as baits in yeast two-hybrid (YTH) assays to screen an RSV cDNA library and were found to interact with the viral RNA-dependent RNA polymerase (RdRp) of RSV. Interactions were confirmed by pull-down and BiFC assays. Further analysis showed that the N-terminus (residues 1-296) of the RdRp was crucial for the interaction between the HSP20s and viral RdRp and responsible for the alteration of the sub-cellular localization and distribution pattern of HSP20s in protoplasts of rice and epidermal cells of Nicotiana benthamiana. This is the first report that a plant virus or a viral protein alters the expression pattern or sub-cellular distribution of sHSPs.

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.

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.

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.

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.

Completion of the sequence of rice gall dwarf virus from Guangxi, China.

The complete nucleotide sequences of segments S2, S3, S5, and S8-S11 of a Rice gall dwarf virus isolate from Guangxi Province, China, (RGDV-GX) were determined, completing the sequence of this isolate. The total genome (25,567nt) was similar in organization to a recently reported Thailand isolate (RGDV-T). A previously unreported second segment-specific repeat of 7 or 8nt was detected close to the 3'-end of segments S3, S5, and S8. The 3'-UTR of RGDV-GX S4 was 139nt shorter than that of RGDV-T; the insertion in RGDV-T contains a 34-nt inverted repeat, with the 3'-terminus probably abolishing the expected stem loop structure.

Molecular characterization of the largest and smallest genome segments, S1 and S12, of Rice gall dwarf virus.

The nucleotide sequences of segments S1 and S12 of a Chinese isolate of Rice gall dwarf virus (RGDV) were determined. This provides the first complete sequences of these segments. The complete sequence of S1, the largest genome segment of RGDV, was 4,505 nucleotides in length and was predicted to encode a large protein of 1,458 amino acids with a calculated molecular mass of nearly 166.2 kDa. The protein was related to that encoded by S1 of Rice dwarf virus (RDV; 50% identity and 67% similarity) and (to a lesser extent) to some large proteins of other reoviruses. It appears to be an RNA-dependent RNA polymerase (RdRp) and is probably present in particles as a minor core protein. S12, the smallest genome segment of RGDV, was 853 nucleotides in length, encoding a single major protein of 206 amino acids with a calculated molecular mass of nearly 23.6 kDa. This protein, though a little larger than those of RDV S11 and Wound tumor virus (WTV) S12 in size, showed some similarity to them, especially in the conserved N-terminal region and may have RNA-binding properties. Despite having a common host plant, RDV and RGDV were not more closely related to one another than either of them was to WTV. Phylogenetic analysis of the RdRp showed that members of the genus Phytoreovirus were more closely related to those of the genus Rotavirus than to any other genus within the family Reoviridae.

Cloning and Sequencing of the Genome Segment S9 of Rice Black-streaked Dwarf Virus.

Genome segment S9's of rice black-streaked dwarf virus (RBSDV) of three Chinese isolates were amplified by RT-PCR and sequenced, and were found to be consisted of 1 900 nt (RBSDV-Zj S9 EMBL accession number AJ297430),1 898 nt (RBSDV-Heb S9 EMBL accession number AJ297429) and 1 900 nt (RBSDV-Hub S9 EMBL accession number AJ291706), respectively. Genome segment S9's of three Chinese Isolates shared 98.5%--98.8% sequence homology and all contained two open reading frames (ORF), which encoded two polypeptides with moleclular weights of 40 kD and 24 kD, respectively. Amino acid sequence comparison of the polypeptides encoded by the second ORF of the corresponding genomic segment of five isolates, including three Chinese isolates, Japanese isolate, and Italian MRDV (maize rough dwarf virus), were highly conserved.