Maize AKINßγ Proteins Interact with P8 of Rice Black Streaked Dwarf Virus and Inhibit Viral Infection.

Rice black streaked dwarf virus (RBSDV) is an important agent causing maize rough dwarf disease, whereas the host factors responding to RBSDV infection are poorly understood. To uncover the molecular interactions between RBSDV and maize, a yeast two-hybrid screen of a maize cDNA library was carried out using the viral P8 protein as a bait. ZmAKINßγ-1 and ZmAKINßγ-2 (ßγ subunit of Arabidopsis SNF1 kinase homolog in maize) possessing high sequence similarities (encoded by two gene copies) were identified as interaction partners. Their interactions with P8 were confirmed in both Nicotiana benthamiana cells and maize protoplasts by bimolecular fluorescence complementation assay. The accumulation levels of ZmAKINßγ mRNAs were upregulated at the stage of the viral symptoms beginning to appear and then downregulated. ZmAKINßγs are putative regulatory subunits of the SnRK1 complex, a core regulator for energy homeostasis. Knockdown of ZmAKINßγs in maize regulated the expression levels of the genes involved in sugar synthesis or degradation, and also the contents of both glucose and sucrose. Importantly, downregulation of ZmAKINßγs expressions facilitated the accumulation of RBSDV in maize. These results implicate a role of ZmAKINßγs in the regulation of primary carbohydrate metabolism, and in the defense against RBSDV infection.

Occurrence and alternation of cytorhabdoviruses on wheat in northern China / Ocorrência e alternância de cytorhabdovirus em trigo no norte da China

Northern cereal mosaic cytorhabdovirus (NCMV) and Barley yellow striate mosaic cytorhabdovirus (BYSMV) are two of the most important viral pathogens of wheat. Northern China is the main wheatproducing region in the country. Wheat growing regions pertaining to four provinces, located in northern China, were surveyed for occurrence of NCMV and BYSMV during the growing seasons of the years 2010 and 2016. Wheat leaf samples were collected randomly from symptomatic plants displaying stunting, chlorotic stripes or mosaic. Roughly 73 samples were collected in the year 2010 from 13 fields, and 154 samples were collected in 2016 from 41 fields. Samples were tested for the presence of NCMV or BYSMV using multiplex reverse transcription-polymerase chain reaction (mRTPCR). The results suggested that BYSMV (49.32% in 2010, 82.47% in 2016) is gradually replacing NCMV (87.67% in 2010, 13.64% in 2016) and becoming the main cytorhabdovirus in different wheat growing regions in northern China.

O cytorhabdovirus do mosaico do cereal do norte (NCMV) e o cytorhabdovirus do mosaico estriado amarelo da cevada (BYSMV) são dois dos mais importantes patógenos virais do trigo. O norte da China é a principal região produtora de trigo do país. As regiões produtoras de trigo pertencentes a quatro províncias do norte da China foram pesquisadas quanto à ocorrência de NCMV e BYSMV durante as safras dos anos de 2010 e 2016. Amostras de folhas de trigo foram coletadas aleatoriamente de plantas sintomáticas, exibindo listras ou mosaico clorótico com baixo crescimento. Cerca de 73 amostras foram coletadas no ano de 2010 a partir de 13 campos, e 154 amostras foram coletadas em 2016 de 41 campos. As amostras foram testadas quanto à presença de NCMV ou BYSMV usando reação em cadeia da polimerase de transcrição reversa multiplex (mRT-PCR). Os resultados sugerem que o BYSMV (49,32% em 2010, 82,47% em 2016) está gradualmente substituindo o NCMV (87,67% em 2010, 13,64% em 2016) e se tornando o principal cytorhabdovirus em diferentes regiões produtoras de trigo no norte da China.

Characterization of small interfering RNAs derived from Rice black streaked dwarf virus in infected maize plants by deep sequencing.

Rice black streaked dwarf virus (RBSDV) is the casual agent of maize rough dwarf disease, which frequently causes severe yield loss in China. However, the interaction between RBSDV and maize plants is largely unknown. RNA silencing is a conserved mechanism against viruses in plants. To understand the antiviral RNA interfering response in RBSDV-infected plants, the profile of virus-derived small interfering RNAs (vsiRNAs) from RBSDV in infected maize plants was obtained by deep sequencing in this study. Our data showed that vsiRNAs, accumulated preferentially as 21- and 22-nucleotide (nt) species, were mapped against all 10 genomic RNA segments of RBSDV and derived almost equally overall from both positive and negative strands, while there were significant differences in the accumulation level of vsiRNAs from segments 2, 4, 6, 7 and 10. The vsiRNAs (21 and 22 nt) generated from each segment of RBSDV genome had a 5'-terminal nucleotide bias toward adenine and uracil. The single-nucleotide resolution maps showed that RBSDV-derived siRNAs preferentially distributed in the 5'- or 3'-terminal regions of several genomic segments. In addition, our results showed that the mRNA levels of some components involved in antiviral RNA silencing pathway were differentially modified during RBSDV infection. Among them, the accumulation levels of ZmDCL1, ZmDCL2, ZmDCL3a, ZmAGO1a, ZmAGO1b, ZmAGO2a, ZmAGO18a and ZmRDR6 mRNAs were significantly up-regulated, while those of ZmDCL3b, ZmDCL4 and ZmAGO1c mRNAs showed no obvious changes in RBSDV-infected maize plants.

Suppression of Jasmonic Acid-Mediated Defense by Viral-Inducible MicroRNA319 Facilitates Virus Infection in Rice.

MicroRNAs (miRNAs) are pivotal modulators of plant development and host-virus interactions. However, the roles and action modes of specific miRNAs involved in viral infection and host susceptibility remain largely unclear. In this study, we show that Rice ragged stunt virus (RRSV) infection caused increased accumulation of miR319 but decreased expression of miR319-regulated TCP (TEOSINTE BRANCHED/CYCLOIDEA/PCF) genes, especially TCP21, in rice plants. Transgenic rice plants overexpressing miR319 or downregulating TCP21 exhibited disease-like phenotypes and showed significantly higher susceptibility to RRSV in comparison with the wild-type plants. In contrast, only mild disease symptoms were observed in RRSV-infected lines overexpressing TCP21 and especially in the transgenic plants overexpressing miR319-resistant TCP21. Both RRSV infection and overexpression of miR319 caused the decreased endogenous jasmonic acid (JA) levels along with downregulated expression of JA biosynthesis and signaling-related genes in rice. However, treatment of rice plants with methyl jasmonate alleviated disease symptoms caused by RRSV and reduced virus accumulation. Taken together, our results suggest that the induction of miR319 by RRSV infection in rice suppresses JA-mediated defense to facilitate virus infection and symptom development.

Characterization of the complete genome of Barley yellow striate mosaic virus reveals a nested gene encoding a small hydrophobic protein.

Barley yellow striate mosaic virus (BYSMV), a member of the genus Cytorhabdovirus, causes serious crop losses in agriculture. Here, we have cloned the BYSMV-derived small interfering RNAs (siRNAs), assembled the siRNAs and used RT-PCR to reconstruct the BYSMV genome. The genome consists of 12,706 nucleotides and encodes ten predicted genes from the antigenomic strand. The major BYSMV structural proteins share identities ranging from 35% to 62% with northern cereal mosaic virus (NCMV) counterparts. A notable difference is that BYSMV contains three transcriptional units residing between the P and M genes compared with four units in the corresponding region of NCMV. Unexpectedly, the middle mRNA in this region encodes gene5 nested in an alternative frame within gene4 via a leaky scanning mechanism. The gene5 encodes a small hydrophobic protein targeting to the endoplasmic reticulum (ER). To our knowledge, this is the first report of nested gene in plant rhabdoviruses.

Selection of reference genes for gene expression studies in virus-infected monocots using quantitative real-time PCR.

Both genome-wide transcriptomic surveys of the mRNA expression profiles and virus-induced gene silencing-based molecular studies of target gene during virus-plant interaction involve the precise estimation of the transcript abundance. Quantitative real-time PCR (qPCR) is the most widely adopted technique for mRNA quantification. In order to obtain reliable quantification of transcripts, identification of the best reference genes forms the basis of the preliminary work. Nevertheless, the stability of internal controls in virus-infected monocots needs to be fully explored. In this work, the suitability of ten housekeeping genes (ACT, EF1α, FBOX, GAPDH, GTPB, PP2A, SAND, TUBß, UBC18 and UK) for potential use as reference genes in qPCR were investigated in five different monocot plants (Brachypodium, barley, sorghum, wheat and maize) under infection with different viruses including Barley stripe mosaic virus (BSMV), Brome mosaic virus (BMV), Rice black-streaked dwarf virus (RBSDV) and Sugarcane mosaic virus (SCMV). By using three different algorithms, the most appropriate reference genes or their combinations were identified for different experimental sets and their effectiveness for the normalisation of expression studies were further validated by quantitative analysis of a well-studied PR-1 gene. These results facilitate the selection of desirable reference genes for more accurate gene expression studies in virus-infected monocots.

Enhanced virus resistance in transgenic maize expressing a dsRNA-specific endoribonuclease gene from E. coli.

Maize rough dwarf disease (MRDD), caused by several Fijiviruses in the family Reoviridae, is a global disease that is responsible for substantial yield losses in maize. Although some maize germplasm have low levels of polygenic resistance to MRDD, highly resistant cultivated varieties are not available for agronomic field production in China. In this work, we have generated transgenic maize lines that constitutively express rnc70, a mutant E. coli dsRNA-specific endoribonuclease gene. Transgenic lines were propagated and screened under field conditions for 12 generations. During three years of evaluations, two transgenic lines and their progeny were challenged with Rice black-streaked dwarf virus (RBSDV), the causal agent of MRDD in China, and these plants exhibited reduced levels of disease severity. In two normal years of MRDD abundance, both lines were more resistant than non-transgenic plants. Even in the most serious MRDD year, six out of seven progeny from one line were resistant, whereas non-transgenic plants were highly susceptible. Molecular approaches in the T12 generation revealed that the rnc70 transgene was integrated and expressed stably in transgenic lines. Under artificial conditions permitting heavy virus inoculation, the T12 progeny of two highly resistant lines had a reduced incidence of MRDD and accumulation of RBSDV in infected plants. In addition, we confirmed that the RNC70 protein could bind directly to RBSDV dsRNA in vitro. Overall, our data show that RNC70-mediated resistance in transgenic maize can provide efficient protection against dsRNA virus infection.

Identification of promoter motifs regulating ZmeIF4E expression level involved in maize rough dwarf disease resistance in maize (Zea Mays L.).

Maize rough dwarf disease (MRDD, a viral disease) results in significant grain yield losses, while genetic basis of which is largely unknown. Based on comparative genomics, eukaryotic translation initiation factor 4E (eIF4E) was considered as a candidate gene for MRDD resistance, validation of which will help to understand the possible genetic mechanism of this disease. ZmeIF4E (orthologs of eIF4E gene in maize) encodes a protein of 218 amino acids, harboring five exons and no variation in the cDNA sequence is identified between the resistant inbred line, X178 and susceptible one, Ye478. ZmeIF4E expression was different in the two lines plants treated with three plant hormones, ethylene, salicylic acid, and jasmonates at V3 developmental stage, suggesting that ZmeIF4E is more likely to be involved in the regulation of defense gene expression and induction of local and systemic resistance. Moreover, four cis-acting elements related to plant defense responses, including DOFCOREZM, EECCRCAH1, GT1GAMSCAM4, and GT1CONSENSUS were detected in ZmeIF4E promoter for harboring sequence variation in the two lines. Association analysis with 163 inbred lines revealed that one SNP in EECCRCAH1 is significantly associated with CSI of MRDD in two environments, which explained 3.33 and 9.04 % of phenotypic variation, respectively. Meanwhile, one SNP in GT-1 motif was found to affect MRDD resistance only in one of the two environments, which explained 5.17 % of phenotypic variation. Collectively, regulatory motifs respectively harboring the two significant SNPs in ZmeIF4E promoter could be involved in the defense process of maize after viral infection. These results contribute to understand maize defense mechanisms against maize rough dwarf virus.

Alteration of gene expression profile in maize infected with a double-stranded RNA fijivirus associated with symptom development.

Maize rough dwarf disease caused by Rice black-streaked dwarf virus (RBSDV) is a major viral disease in China. It has been suggested that the viral infection of plants might cause distinct disease symptoms through the inhibition or activation of host gene transcription. We scanned the gene expression profile of RBSDV-infected maize through oligomer-based microarrays to reveal possible expression changes associated with symptom development. Our results demonstrate that various resistance-related maize genes and cell wall- and development-related genes, such as those for cellulose synthesis, are among the genes whose expression is dramatically altered. These results could aid in research into new strategies to protect cereal crops against viruses, and reveal the molecular mechanisms of development of specific symptoms in rough dwarf-related diseases.

Complete genomic sequence analysis of a highly virulent isolate revealed a novel strain of Sugarcane mosaic virus.

Sugarcane mosaic virus (SCMV) is the most prevalent virus causing maize dwarf mosaic disease in northern China. A SCMV isolate, BD8, was obtained from the maize showing dwarf and mosaic symptoms in Baoding, China. The complete genomic sequence of BD8 is 9,576 nucleotides (nt) excluding the poly(A) tail. It contains one single open reading frame of 9,192 nt and encodes a large polyprotein of 3,063 amino acids (aa), flanked by a 5'-untranslated region (UTR) of 148 nt and a 3'-UTR of 236 nt. The entire genomic sequence of BD8 shares identities of 79.1-80.8% with those of other 13 SCMV isolates available in the GenBank at nt level, while their CP genes share identities of 76.9-82.6 and 82.8-86.9% at nt and aa levels, respectively. Phylogenetic analysis of the complete genomic sequences reveals that SCMV can be clustered to four groups: group I includes isolates from maize, group II consists of isolates from sugarcane or maize, groups III and IV contain single isolate of AU-A (AJ278405) and BD8, respectively. Thus, BD8 represents a new strain of SCMV. Furthermore analysis of the CP gene sequences of more isolates shows that BD8 is clustered to a group with the isolates from Thailand and Vietnam, which implies that isolates of this strain have been distributed in South Asia. In the greenhouse, BD8 can cause severe symptoms in all the 12 maize varieties tested with high incidence, indicating that BD8 is highly virulent.

Rice black streaked dwarf virus P9-1, an alpha-helical protein, self-interacts and forms viroplasms in vivo.

Replication and assembly of viruses from the family Reoviridae are thought to take place in discrete cytoplasmic inclusion bodies, commonly called viral factories or viroplasms. Rice black streaked dwarf virus (RBSDV) P9-1, a non-structural protein, has been confirmed to accumulate in these intracellular viroplasms in infected plants and insects. However, little is known about its exact function. In this study, P9-1 of RBSDV-Baoding was expressed in Escherichia coli as a His-tagged fusion protein and analysed using biochemical and biophysical techniques. Mass spectrometry and circular dichroism spectroscopy studies showed that P9-1 was a thermostable, alpha-helical protein with a molecular mass of 41.804 kDa. A combination of gel-filtration chromatography, chemical cross-linking and a yeast two-hybrid assay was used to demonstrate that P9-1 had the intrinsic ability to self-interact and form homodimers in vitro and in vivo. Furthermore, when transiently expressed in Arabidopsis protoplasts, P9-1 formed large, discrete viroplasm-like structures in the absence of infection or other RBSDV proteins. Taken together, these results suggest that P9-1 is the minimal viral component required for viroplasm formation and that it plays an important role in the early stages of the virus life cycle by forming intracellular viroplasms that serve as the sites of virus replication and assembly.