Xin Shun Ding

Characterization of a Brome mosaic virus Strain and Its Use as a Vector for Gene Silencing in Monocotyledonous Hosts

Virus-induced gene silencing (VIGS) is used to analyze gene function in dicotyledonous plants but less so in monocotyledonous plants (particularly rice and corn), partially due to the limited number of virus expression vectors available. Here, we report the cloning and modification for VIGS of a virus from Festuca arundinacea Schreb. (tall fescue) that caused systemic mosaic symptoms on barley, rice, and a specific cultivar of maize (Va35) under greenhouse conditions. Through sequencing, the virus was determined to be a strain of Brome mosaic virus (BMV). The virus was named F-BMV (F for Festuca), and genetic determinants that controlled the systemic infection of rice were mapped to RNAs 1 and 2 of the tripartite genome. cDNA from RNA 3 of the Russian strain of BMV (R-BMV) was modified to accept inserts from foreign genes. Coinoculation of RNAs 1 and 2 from F-BMV and RNA 3 from R-BMV expressing a portion of a plant gene to leaves of barley, rice, and maize plants resulted in visual silencing-like phenotypes. The visual phenotypes were correlated with decreased target host transcript levels in the corresponding leaves. The VIGS visual phenotype varied from maintained during silencing of actin 1 transcript expression to transient with incomplete penetration through affected tissue during silencing of phytoene desaturase expression. F-BMV RNA 3 was modified to allow greater accumulation of virus while minimizing virus pathogenicity. The modified vector C-BMV(A/G) (C for chimeric) was shown to be useful for VIGS. These BMV vectors will be useful for analysis of gene function in rice and maize for which no VIGS system is reported.

The Tobacco mosaic virus 126-kDa Protein Associated with Virus Replication and Movement Suppresses RNA Silencing

Systemic symptoms induced on Nicotiana tabacum cv. Xanthi by Tobacco mosaic virus (TMV) are modulated by one or both amino-coterminal viral 126- and 183-kDa proteins: proteins involved in virus replication and cell-to-cell movement. Here we compare the systemic accumulation and gene silencing characteristics of TMV strains and mutants that express altered 126- and 183-kDa proteins and induce varying intensities of systemic symptoms on N. tabacum. Through grafting experiments, it was determined that M(IC)1,3, a mutant of the masked strain of TMV that accumulated locally and induced no systemic symptoms, moved through vascular tissue but failed to accumulate to high levels in systemic leaves. The lack of M(IC)1,3 accumulation in systemic leaves was correlated with RNA silencing activity in this tissue through the appearance of virus-specific, approximately 25-nucleotide RNAs and the loss of fluorescence from leaves of transgenic plants expressing the 126-kDa protein fused with green fluorescent protein (GFP). The ability of TMV strains and mutants altered in the 126-kDa protein open reading frame to cause systemic symptoms was positively correlated with their ability to transiently extend expression of the 126-kDa protein:GFP fusion and transiently suppress the silencing of free GFP in transgenic N. tabacum and transgenic N. benthamiana, respectively. Suppression of GFP silencing in N. benthamiana occurred only where virus accumulated to high levels. Using agroinfiltration assays, it was determined that the 126-kDa protein alone could delay GFP silencing. Based on these results and the known synergies between TMV and other viruses, the mechanism of suppression by the 126-kDa protein is compared with those utilized by other originally characterized suppressors of RNA silencing.

Molecular cloning and characterisation of a venom allergen AG5-like cDNA from Meloidogyne incognita

RNA fingerprinting was used to identify RNAs that were expressed in parasitic second-stage juveniles of Meloidogyne incognita, but absent from or reduced in preparasitic second-stage juveniles. A cDNA encoding a putative secretory protein was cloned from a M. incognita second-stage juvenile cDNA library by probing with a 0.5kb fragment derived from fingerprinting that was more strongly expressed in parasitic second-stage juveniles. The cDNA, named Mi-msp-1, contained an open reading frame encoding 231 amino acids, with the first 21 amino acids being a putative secretory signal. In Southern blot analysis the Mi-msp-1 hybridised with genomic DNA from M. incognita, Meloidogyne arenaria, Meloidogyne javanica, but not Meloidogyne hapla, Heterodera glycines or Caenorhabditis elegans. In Northern blot analysis a 1kb transcript was detected in both preparasitic and parasitic second-stage juveniles, but not in adult females of M. incognita. Comparing the predicted amino acid sequence with protein databases revealed significant similarity to the venom allergen antigen 5 family of proteins in hymenoptera insects and homologues found in several other nematode species.

Development of Tobacco Mosaic Virus Infection Sites in Nicotiana benthamiana

To monitor infection of Nicotiana benthamiana by tobacco mosaic virus (TMV), leaves were inoculated with viral constructs expressing the green fluorescent protein (GFP) from jellyfish (Aequorea victoria) fused to the movement protein (MP) of TMV (MP:GFP) or as a free GFP in place of the coat protein (CP). Infection sites produced by TMV expressing the MP:GFP appeared as fluorescent rings larger in diameter and less fluorescent than fluorescent disks induced by constructs encoding free GFP. These results suggest that protein expression driven by the MP subgenomic promoter (sgp) initiates and ends earlier and is at lower level than that observed for proteins driven by the CP sgp. Similarly, analyses of cross sections through the infection sites revealed that in different cell types the accumulation of MP:GFP was regulated differently than the accumulation of free GFP. Immunocytochemistry and electron microscopy showed that near the leading edge of the fluorescent ring the MP:GFP and the viral 126 kDa and 18...

Functions of rice NAC transcriptional factors, ONAC122 and ONAC131, in defense responses against Magnaporthe grisea.

NAC (NAM/ATAF/CUC) transcription factors have important functions in regulating plant growth, development, and abiotic and biotic stress responses. Here, we characterized two rice pathogen-responsive NAC transcription factors, ONAC122 and ONAC131. We determined that these proteins localized to the nucleus when expressed ectopically and had transcriptional activation activities. ONAC122 and ONAC131 expression was induced after infection by Magnaporthe grisea, the causal agent of rice blast disease, and the M. grisea-induced expression of both genes was faster and higher in the incompatible interaction compared with the compatible interaction during early stages of infection. ONAC122 and ONAC131 were also induced by treatment with salicylic acid, methyl jasmonate or 1-aminocyclopropane-1-carboxylic acid (a precursor of ethylene). Silencing ONAC122 or ONAC131 expression using a newly modified Brome mosaic virus (BMV)-based silencing vector resulted in an enhanced susceptibility to M. grisea. Furthermore, expression levels of several other defense- and signaling-related genes (i.e. OsLOX, OsPR1a, OsWRKY45 and OsNH1) were down-regulated in plants silenced for ONAC122 or ONAC131 expression via the BMV-based silencing system. Our results suggest that both ONAC122 and ONAC131 have important roles in rice disease resistance responses through the regulated expression of other defense- and signaling-related genes.

Infection of Barley by Brome Mosaic Virus Is Restricted Predominantly to Cells in and Associated with Veins through a Temperature-Dependent Mechanism

Results from previous cytological studies on barley (Hordeum vulgare) infected with brome mosaic virus (BMV) indicated that this virus can infect and accumulate to high levels in mesophyll and other cell types within the leaves. Through immunocytochemistry and in situ hybridization, we have determined that BMV infection in barley is restricted predominantly to cells within and associated with vasculature when plants are grown at 24/20°C (day/night). This tissue restriction can be fully overcome by growing infected plants at 34°C for 2 h. Our results also indicate that BMV is likely to start systemic infection of young, uninoculated leaves in barley before spreading into all longitudinal veins in inoculated leaves. Possible barrier(s) to BMV movement between vascular bundle sheath cells and mesophyll cells, and the relationship between virus and photoassimilate transport through longitudinal and transverse veins are discussed.

Analysis of Gene Function in Rice Through Virus-Induced Gene Silencing

Virus-induced gene silencing (VIGS) is a powerful RNA-silencing based technology adapted for the study of host-gene function. VIGS functions through the expression of a host gene from a virus vector. Both the virus-encoded host sequence and the homologous host target messenger RNA are destroyed or made inactive through a host surveillance system. Here, we describe procedures for the use of a new virus vector for VIGS in monocotyledonous hosts and, in particular, in rice (Oryza sativa), a species for which no VIGS vector was previously available.

Maize Elongin C interacts with the viral genome‐linked protein, VPg, of Sugarcane mosaic virus and facilitates virus infection

The viral genome-linked protein, VPg, of potyviruses is involved in viral genome replication and translation. To determine host proteins that interact with Sugarcane mosaic virus (SCMV) VPg, a yeast two-hybrid screen was used and a maize (Zea mays) Elongin C (ZmElc) protein was identified. ZmELC transcript was observed in all maize organs, but most highly in leaves and pistil extracts, and ZmElc was present in the cytoplasm and nucleus of maize cells in the presence or absence of SCMV. ZmELC expression was increased in maize tissue at 4 and 6 d post SCMV inoculation. When ZmELC was transiently overexpressed in maize protoplasts the accumulation of SCMV RNA was approximately doubled compared with the amount of virus in control protoplasts. Silencing ZmELC expression using a Brome mosaic virus-based gene silencing vector (virus-induced gene silencing) did not influence maize plant growth and development, but did decrease RNA accumulation of two isolates of SCMV and host transcript encoding ZmeIF4E during SCMV infection. Interestingly, Maize chlorotic mottle virus, from outside the Potyviridae, was increased in accumulation after silencing ZmELC expression. Our results describe both the location of ZmElc expression in maize and a new activity associated with an Elc: support of potyvirus accumulation.

Rationale for Developing New Virus Vectors to Analyze Gene Function in Grasses Through Virus-Induced Gene Silencing

The exploding availability of genome and EST-based sequences from grasses requires a technology that allows rapid functional analysis of the multitude of genes that these resources provide. There are several techniques available to determine a genes function. For gene knockdown studies, silencing through RNAi is a powerful tool. Gene silencing can be accomplished through stable transformation or transient expression of a fragment of a target gene sequence. Stable transformation in rice, maize, and a few other species, although routine, remains a relatively low-throughput process. Transformation in other grass species is difficult and labor-intensive. Therefore, transient gene silencing methods including Agrobacterium-mediated and virus-induced gene silencing (VIGS) have great potential for researchers studying gene function in grasses. VIGS in grasses already has been used to determine the function of genes during pathogen challenge and plant development. It also can be used in moderate-throughput reverse genetics screens to determine gene function. However, the number of viruses modified to serve as silencing vectors in grasses is limited, and the silencing phenotype induced by these vectors is not optimal: the phenotype being transient and with moderate penetration throughout the tissue. Here, we review the most recent information available for VIGS in grasses and summarize the strengths and weaknesses in current virus-grass host systems. We describe ways to improve current virus vectors and the potential of other grass-infecting viruses for VIGS studies. This work is necessary because VIGS for the foreseeable future remains a higher throughput and more rapid system to evaluate gene function than stable transformation.

Presence of Brome mosaic virus in Barley Guttation Fluid and Its Association with Localized Cell Death Response

ABSTRACT Water exits from inside the leaf through transpiration or guttation. Under conditions to promote guttation, surface fluid (guttation fluid) from Brome mosaic virus (BMV)-infected barley, wheat, and maize plants was analyzed for the presence of the virus by biological and serological assays. We also investigated the route by which BMV exited infected cells to the intercellular space of the barley leaf. BMV was detected in guttation fluid from systemically infected barley leaves when the initial viral symptoms were observed on these leaves. The virus was also detected in guttation fluid from systemically infected wheat leaves, but not in maize leaves showing either systemic necrosis or chlorotic streaks. Interestingly, in BMV-infected barley leaves, but not in maize leaves showing chlorotic streaks, cell death occurred within and adjacent to veins. Staining of xylem and phloem networks in infected barley leaves with fluorescent dyes showed that xylem, and to a lesser extent phloem, were severely damaged and thus became leaky for dye transport. No such damage was observed in BMV-infected maize leaves showing chlorotic streaks. We propose that in infected barley leaves, BMV exits from damaged vein cells (especially the xylem elements), accumulates in intercellular spaces, and then reaches the surface of the leaves through stomata during guttation or transpiration. In nature, BMV may be carried to adjacent plants and cause infection by movement of vertebrate and invertebrate vectors among infected plants exuding guttation fluid.