Brachypodium Phenylalanine Ammonia Lyase (PAL) Promotes Antiviral Defenses against Panicum mosaic virus and Its Satellites.

Brachypodium distachyon has recently emerged as a premier model plant for monocot biology, akin to Arabidopsis thaliana We previously reported genome-wide transcriptomic and alternative splicing changes occurring in Brachypodium during compatible infections with Panicum mosaic virus (PMV) and its satellite virus (SPMV). Here, we dissected the role of Brachypodium phenylalanine ammonia lyase 1 (PAL1), a key enzyme for phenylpropanoid and salicylic acid (SA) biosynthesis and the induction of plant defenses. Targeted metabolomics profiling of PMV-infected and PMV- plus SPMV-infected (PMV/SPMV) Brachypodium plants revealed enhanced levels of multiple defense-related hormones and metabolites such as cinnamic acid, SA, and fatty acids and lignin precursors during disease progression. The virus-induced accumulation of SA and lignin was significantly suppressed upon knockdown of B. distachyonPAL1 (BdPAL1) using RNA interference (RNAi). The compromised SA accumulation in PMV/SPMV-infected BdPAL1 RNAi plants correlated with weaker induction of multiple SA-related defense gene markers (pathogenesis related 1 [PR-1], PR-3, PR-5, and WRKY75) and enhanced susceptibility to PMV/SPMV compared to that of wild-type (WT) plants. Furthermore, exogenous application of SA alleviated the PMV/SPMV necrotic disease phenotypes and delayed plant death caused by single and mixed infections. Together, our results support an antiviral role for BdPAL1 during compatible host-virus interaction, perhaps as a last resort attempt to rescue the infected plant.IMPORTANCE Although the role of plant defense mechanisms against viruses are relatively well studied in dicots and in incompatible plant-microbe interactions, studies of their roles in compatible interactions and in grasses are lagging behind. In this study, we leveraged the emerging grass model Brachypodium and genetic resources to dissect Panicum mosaic virus (PMV)- and its satellite virus (SPMV)-compatible grass-virus interactions. We found a significant role for PAL1 in the production of salicylic acid (SA) in response to PMV/SPMV infections and that SA is an essential component of the defense response preventing the plant from succumbing to viral infection. Our results suggest a convergent role for the SA defense pathway in both compatible and incompatible plant-virus interactions and underscore the utility of Brachypodium for grass-virus biology.

Colony establishment and maintenance of the eriophyid wheat curl mite Aceria tosichella for controlled transmission studies on a new virus-like pathogen.

High plains disease (HPD) is of serious economic concern for wheat and corn production, but little is known about the virus-like causal agent. In the field, HPD is often associated with Wheat streak mosaic virus (WSMV) and both pathogens are transmitted by the same eriophyid wheat curl mite, Aceria tosichella Keifer. The objective of this study was to develop methods for establishing and maintaining HPD-transmitting wheat curl mite colonies for their use in studies on HPD. Towards this goal, mite colonies from a mixed infection source were separated into colonies either (i). not viruliferous; (ii). only transmitting WSMV; or (iii). only transmitting HPD. Maintenance of these colonies required strictly separated incubator facilities and adaptation of mite-suitable transfer techniques to permit frequent passages of mites to healthy plants. The established colonies provided reliable sources of infective material to study the progression of HPD and/or WSMV in plants using sensitive immuno-detection assays. In conclusion, we have developed reliable methods with a poorly studied arthropod vector to examine the biology and properties of a new virus-like disease.

Defective interfering RNAs of a satellite virus.

Panicum mosaic virus (PMV) is a recently molecularly characterized RNA virus with the unique feature of supporting the replication of two subviral RNAs in a few species of the family Gramineae. The subviral agents include a satellite RNA (satRNA) that is devoid of a coding region and the unrelated satellite panicum mosaic virus (SPMV) that encodes its own capsid protein. Here we report the association of this complex with a new entity in the RNA world, a defective-interfering RNA (DI) of a satellite virus. The specificity of interactions governing this four-component viral system is illustrated by the ability of the SPMV DIs to strongly interfere with the accumulation of the parental SPMV. The SPMV DIs do not interfere with PMV satRNA, but they do slightly enhance the rate of spread and titer of PMV. The SPMV-derived DIs provide an additional avenue by which to investigate fundamental biological questions, including the evolution and interactions of infectious RNAs.

Genetic identification of multiple biological roles associated with the capsid protein of satellite panicum mosaic virus.

Satellite panicum mosaic virus (SPMV), an 824-nucleotide, positive-sense, single-stranded RNA virus, depends on Panicum mosaic virus (PMV) for replication and spread in host plants. Compared with PMV infection alone, symptoms are intensified and develop faster on millet plants infected with SPMV and PMV. SPMV encodes a 157 amino acid capsid protein (CP) (17.5 kDa) to encapsidate SPMV RNA and form T = 1 satellite virions. The present study identifies additional biological activities of the SPMV CP, including the induction of severe chlorosis on proso millet plants (Panicum miliaceum cv. Sunup or Red Turghai). Initial deletion mutagenesis experiments mapped the chlorosis-inducing domain to amino acids 50 to 157 on the C-terminal portion of the SPMV CP. More defined analyses revealed that amino acids 124 to 135 comprised a critical domain associated with chlorosis induction and virion formation, whereas the extreme C-terminal residues 148 to 157 were not strictly essential for either role. The results also demonstrated that the absence of SPMV CP tended to stimulate the accumulation of defective RNAs. This suggests that the SPMV CP plays a significant role in maintaining the structural integrity of the full-length satellite virus RNA and harbors multiple functions associated with pathogenesis in SPMV-infected host plants.

Complete nucleotide sequence and genome organization of Beet soilborne mosaic virus, a proposed member of the genus Benyvirus.

The complete nucleotide sequences of RNAs 1 to 4 of Beet soilborne mosaic virus (BSBMV) were determined. The genomic organization of BSBMV is identical to Beet necrotic yellow vein virus (BNYVV), the type species of the genus Benyvirus. BSBMV RNA1 encodes a single large open reading frame (ORF) with similar replicase-associated motifs identified for BNYVV. BSBMV RNA2 has six potential ORFs with an organization resembling BNYVV RNA2. RNA3 and RNA4 resemble the analogous BNYVV RNAs, which encode proteins associated with symptom development and fungal transmission, respectively. The predicted ORFs on BNYVV and BSBMV reveal 23% to 83% amino acid identity and the overall nucleotide sequences are 35% to 77% identical. Based on sequence analyses, BSBMV is a new benyvirus that can be distinguished from BNYVV.

RNA: protein interactions associated with satellites of panicum mosaic virus.

The interactions between satellite panicum mosaic virus (SPMV) capsid protein (CP) and its 824 nucleotide (nt) single stranded RNA were investigated by gel mobility shift assay and Northwestern blot assay. SPMV CP has specificity for its RNA at high affinity, but little affinity for non-viral RNA. The SPMV CP also bound a 350 nt satellite RNA (satRNA) that, like SPMV, is dependent on panicum mosaic virus for its replication. SPMV CP has the novel property of encapsidating SPMV RNA and satRNA. Competition gel mobility shift assays performed with a non-viral RNA and unlabeled SPMV RNA and satRNA revealed that these RNA:protein interactions were in part sequence specific.

In vitro- and in vivo-generated defective RNAs of satellite panicum mosaic virus define cis-acting RNA elements required for replication and movement.

Satellite panicum mosaic virus (SPMV) depends on its helper virus, panicum mosaic virus (PMV), to provide trans-acting proteins for replication and movement. The 824-nucleotide (nt) genome of SPMV possesses an open reading frame encoding a 17.5-kDa capsid protein (CP), which is shown to be dispensable for SPMV replication. To localize cis-acting RNA elements required for replication and movement, a comprehensive set of SPMV cDNA deletion mutants was generated. The results showed that the 263-nt 3' untranslated region (UTR) plus 73 nt upstream of the CP stop codon and the first 16 nt in the 5' UTR are required for SPMV RNA amplification and/or systemic spread. A region from nt 17 to 67 within the 5' UTR may have an accessory role in RNA accumulation, and a fragment bracketing nt 68 to 104 appears to be involved in the systemic movement of SPMV RNA in a host-dependent manner. Unexpectedly, defective RNAs (D-RNAs) accumulated de novo in millet plants coinfected with PMV and either of two SPMV mutants: SPMV-91, which is incapable of expressing the 17.5-kDa CP, and SPMV-GUG, which expresses low levels of the 17.5-kDa CP. The D-RNA derived from SPMV-91 was isolated from infected plants and used as a template to generate a cDNA clone. RNA transcripts derived from this 399-nt cDNA replicated and moved in millet plants coinoculated with PMV. The characterization of this D-RNA provided a biological confirmation that the critical RNA domains identified by the reverse genetic strategy are essential for SPMV replication and movement. The results additionally suggest that a potential "trigger" for spontaneous D-RNA accumulation may be associated with the absence or reduced accumulation of the 17.5-kDa SPMV CP. This represents the first report of a D-RNA associated with a satellite virus.

cDNA probes for detection of specific dsRNAs from the fungal pathogen, Monosporascus cannonballus.

Monosporascus cannonballus is an ascomycete fungus that is the causative agent of Monosporascus root rot/vine decline, a serious disease of muskmelon and watermelon. Double-stranded RNA (dsRNA) was identified in approximately 60% of M. cannonballus isolates recovered from infected muskmelon plants in 1993. After repeated laboratory transfer on culture media, the majority of the isolates harboring dsRNAs developed degenerate culture phenotypes and showed reduced virulence (hypovirulence) to muskmelon. Initially, dsRNA purification and cDNA synthesis were attempted in three M. cannonballus isolates harboring dsRNAs. However, numerous difficulties were encountered due to the stable, double-stranded nature of the dsRNAs and contamination of the preparations by fungal rRNA. Several purification and cDNA protocols were evaluated and eventually modified into methods that were ultimately highly effective for cloning dsRNAs from M. cannonballus. The cDNAs derived from purified dsRNA preparations were cloned into a pUC119 plasmid vector and amplified in Escherichia coli. Nine cDNA clones were identified that are specific for medium-sized (ca. 3 kbp) dsRNAs associated with M. cannonballus isolate Ca91-17(96+). The methods used to make the cDNA clones of the dsRNAs in M. cannonballus may be useful for those working on fungal dsRNAs. In addition, these cDNAs may be useful for identifying dsRNAs associated with the hypovirulence phenotype.

Genetic Diversity of Panicum mosaic virus Satellite RNAs in St. Augustinegrass.

ABSTRACT St. Augustine decline is a viral disease caused by Panicum mosaic virus (PMV) alone or in combination with a satellite virus (SPMV) and/or satellite RNAs (satRNAs). A ribonuclease protection assay (RPA) was used to evaluate the genetic diversity of PMV satRNAs isolated from 100 naturally infected St. Augustinegrass plants (Stenotaphrum secundatum). Distinctive satRNA RPA profiles were observed for 40 of 52 samples from College Station (CS) and 37 of 48 samples from Corpus Christi (CC), Texas. A dendrogram constructed from the RPA data revealed that satRNAs were grouped in two distinct clusters based on their place of origin. From 100 samples, only 4 satRNAs from CS were placed in the CC group, and only 2 satRNAs from CC were placed in the CS group. The data show that there is genetic variability in PMV satRNAs in naturally occurring infections, and distinct geographically separate populations can be identified from CC and CS.

A gene cluster encoded by panicum mosaic virus is associated with virus movement.

A subgenomic RNA (sgRNA) of about 1500 nucleotides has been detected in millet plants and protoplasts infected with panicum mosaic virus (PMV). This sgRNA expressed p8, p6.6, p15, and the 26-kDa capsid protein (CP) genes during in vitro translation assays, as determined by using mutants inactivated for expression of each open reading frame. Abolishing expression of p8 and p6.6, the two 5'-proximal genes on the sgRNA, did not affect the replication of PMV in millet protoplasts, but obstructed spread in plants. As predicted for a typical cell-to-cell movement protein, p8 localized to the cell wall fraction of PMV-infected millet plants. The introduction of premature stop codons downstream of the PMV p15 start codon (p15*) abolished infectivity in planta, but did not impair replication in protoplasts. However, a delayed systemic infection in millet plants was supported by the p15aug(-) start codon mutant, which may reflect very low levels of expression from a suboptimal start codon context and/or leaky scanning to a second inframe AUG codon to express the C-terminal portion of the 15-kDa protein. PMV CP mutants had little effect on sgRNA accumulation, but were correlated with a reduction of the gRNA and the decreased expression of the 8-kDa protein in protoplasts as well as abolishment of cell-to-cell spread in plants. These results imply that the successful establishment of a PMV systemic infection in millet host plants appears to be dependent on the concerted expression of the p8, p6.6, p15, and CP genes.

A New Virus on Maize in Nigeria: Maize Mild Mottle Virus.

Maize (Zea mays) and itch grass (Rottboellia cochinchinensis) plants exhibiting a mild mosaic or mottle were collected from a farmer's field near Mokwa, Nigeria, in 1993. Icosahedral virions (approximately 28 to 30 nm) were purified from symptomatic tissue by differential centrifugation in 0.1 M phosphate buffer, pH 7.0. The virions are composed of one single-stranded positive-sense RNA of approximately 4,000 nucleotides and, as estimated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a capsid protein of approximately 28 kDa. The virus was readily detected in infected plants by enzyme-linked immunosorbent assay and immunoblot assays with a polyclonal rabbit antibody derived from purified virions. A partial cDNA library was generated with random primers. Sequence analyses of a cDNA clone representing a portion of the putative replicase gene aligned most closely with related sequences of viruses within the Tombusviridae. In particular, a region of 78 predicted amino acids surrounding the "GDD" replicase motif shares 73% identity with panicum mosaic virus and 61% identity with maize chlorotic mottle virus. The virus is readily transmitted by mechanical inoculation to sweet and dent corn, millet, and wheat. Currently it is not considered of economic importance in Nigeria. The data suggest that "maize mild mottle virus" is a newly identified virus infecting maize.

Nucleotide sequence and infectivity of a full-length cDNA clone of panicum mosaic virus.

The sequence of an infectious cDNA clone of panicum mosaic virus (PMV) showed that the single-stranded RNA genome is 4326 nucleotides (nt) and a single highly abundant subgenomic (sg) RNA of 1475 nt was synthesized during PMV infection of pearl millet plants and protoplasts. Computer comparisons revealed strong similarities between the predicted amino acid sequences of the p48 and p112 open reading frames (ORFs) and replicase proteins of members of the Tombusviridae. The sgRNA has the potential to encode five proteins. Three small ORFs, p8, p8-FS, and/or p6.6 have similarity to ORFs of carmo-, necro-, and machlomoviruses thought to be involved in virus spread in plants. The sgRNA also has the potential to encode a 26-kDa capsid protein and a 15-kDa nested gene (p15) of unknown function. PMV transcripts also supported replication and movement of SPMV, the satellite virus. Genome organization, physicochemical properties, and biological features indicate that PMV is a member of the Tombusviridae family. However, PMV differs sufficiently from previously described members to warrant its placement in a new genus provisionally designated Panicovirus.

Plant virus gene vectors for transient expression of foreign proteins in plants.

The development of plant virus gene vectors for expression of foreign genes in plants provides attractive biotechnological tools to complement conventional breeding and transgenic methodology. The benefits of virus-based transient RNA and DNA replicons versus transgenic gene expression include rapid and convenient engineering coupled with flexibility for expeditious application in various plant species. These characteristics are especially advantageous when very high levels of gene expression are desired within a short time, although instability of the foreign gene in the viral genome can present some problems. The strategies that have been tested for foreign gene expression in various virus-based vectors include gene replacement, gene insertion, epitope presentation, use of virus controlled gene expression cassettes, and complementation. Recent reports of the utilization of virus vectors for foreign gene expression in fundamental research and biotechnology applications are discussed.

Tomato bushy stunt virus spread is regulated by two nested genes that function in cell-to-cell movement and host-dependent systemic invasion.

We have investigated the importance of two small nested genes (p19 and p22) located near the 3' end of the genome of tomato bushy stunt virus (TBSV) for infectivity in several hosts. Our results show that both genes are dispensable for replication and transcription and that the p19 gene encodes a soluble protein, whereas the p22 gene specifies a membrane-associated protein. Assays using TBSV derivatives that have the beta-glucuronidase gene substituted for the capsid protein gene demonstrate that p22 is required for cell-to-cell movement in all plants tested. Mutations inactivating p19 ameliorate the severe necrotic systemic symptoms elicited by wild-type TBSV in Nicotiana benthamiana and Nicotiana clevelandii, but p19 does not obviously affect movement in these hosts. However, in some local lesion hosts p19 influences the lesion diameter, which suggests that it has an auxiliary host-dependent role in movement. This notion is supported by the observation that p19 is required for long-distance spread of TBSV in spinach and for systemic infection of pepper plants. Thus, movement of TBSV is regulated by two nested genes; p22 governs cell-to-cell movement and p19 has a host-specific role in systemic invasion.

Sequences of the cDNAs encoding the heavy- and light-chain Fab region of an antibody to the phenylurea herbicide diuron.

The cDNAs from a hybridoma (mAb 481.1) specific for diuron, a widely used phenylurea herbicide, were cloned into the phage display vector pComb8. Antigen-binding clones were selected by panning on diuron-hapten-BSA conjugates. The nucleotide and deduced amino-acid sequences encoding the Fab regions of the light (kappa) and heavy (gamma) chains were determined. The light chain was from mouse kappa chain subgroup III and the heavy chain was a member of the mouse H chain subgroup III(d).

Identification of tomato bushy stunt virus host-specific symptom determinants by expression of individual genes from a potato virus X vector.

In this study, we analyzed the influence of two nested genes (p19 and p22) of tomato bushy stunt virus (TBSV) on disease symptoms in systemically infected plants and in local lesion hosts. The contribution of individual genes was determined by bioassays with an infectious clone of wild-type TBSV, with p19/p22 mutant derivatives, and by expression of individual TBSV genes from a heterologous potato virus X (PVX) vector. Our results showed that TBSV genes could be expressed at high levels from the PVX vector. The subcellular localization of these proteins as well as the ability of PVX-expressed p22 to trans complement TBSV cell-to-cell movement defective mutants indicate that the exogenously expressed proteins are functionally active. Inoculation studies with TBSV mutants and the PVX derivatives demonstrated that p19 induced a generalized necrosis upon systemic infection of Nicotiana benthamiana and N. clevelandii. In addition, p19 elicited the formation of local necrotic lesions in N. tabacum; however, in N. glutinosa and N. edwardsonii, the local lesion response was activated by p22. These results show that the p19 and p22 proteins of TBSV are important symptom determinants and that closely related plant species may contain different resistance genes that selectively respond to individual TBSV proteins.