RNA Interference Past and Future Applications in Plants.

Antisense RNA was observed to elicit plant disease resistance and post-translational gene silencing (PTGS). The universal mechanism of RNA interference (RNAi) was shown to be induced by double-stranded RNA (dsRNA), an intermediate produced during virus replication. Plant viruses with a single-stranded positive-sense RNA genome have been instrumental in the discovery and characterization of systemic RNA silencing and suppression. An increasing number of applications for RNA silencing have emerged involving the exogenous application of dsRNA through spray-induced gene silencing (SIGS) that provides specificity and environmentally friendly options for crop protection and improvement.

Impact of Foliar Application of Acibenzolar-S-Methyl on Rose Rosette Disease and Rose Plant Quality.

Rose rosette disease (RRD) caused by rose rosette emaravirus (RRV) is a major issue in the U.S. rose industry with no effective method for its management. This study evaluated the effect of foliar application of acibenzolar-S-methyl (ASM), a plant systemic acquired resistance inducer, in reducing RRD disease severity on Rosa species cv. Radtkopink ('Pink Double Knock Out') under greenhouse conditions, and the effect of ASM on plant growth under commercial nursery production conditions. ASM at 50- or 100-mg/liter concentrations at weekly intervals significantly reduced RRD severity compared with the untreated control in two of the three greenhouse trials (P < 0.05). The plants in these trials were subsequently pruned and observed for symptoms, which further indicated that application of ASM at 50- or 100-mg/liter concentrations lowered disease severity compared with the untreated control (P < 0.05) in these two trials. Plants treated with ASM at 50- or 100-mg/liter concentrations had delayed incidence of RRD compared with the nontreated controls. Plants treated with ASM at the 50- or 100-mg/liter rate in all three trials either did not have RRV present or the virus was present in fewer leaf samples than untreated controls as indicated by quantitative reverse transcription PCR analysis. Overall, plants treated with ASM at the 50-mg/liter concentration had 36 to 43% reduced RRD incidence compared with the water control. The treatment of two cultivars of rose, 'Radtkopink' and 'Meijocos' ('Pink Drift'), with weekly foliar applications of ASM at the three rates (0.5, 0.75, and 1.0 oz/A) indicated that ASM had no negative effect on flowering or plant growth at even the highest rate of application.

A rapid fluorescence-based real-time isothermal assay for the detection of Cucurbit yellow stunting disorder virus in squash and watermelon plants.

Cucurbit yellow stunting disorder virus (CYSDV) is a single-stranded positive-sense RNA virus that produces devastating disease in watermelon and squash. Foliar symptoms of CYSDV consist of interveinal yellowing, brittleness, and thickening of older leaves leading to reduced plant vigor. A rapid diagnostic method for CYSDV would facilitate early detection and implementation of best viral-based management practices. We developed a rapid isothermal reverse transcription-recombination polymerase amplification (exo RT-RPA) assay for the detection of CYSDV. The primers and a 6-fluorescein amidite (6-FAM) probe were developed to target the nucleocapsid gene. The real-time assay detected CYSDV at 2.5 pg purified total RNA extracted from CYSDV-infected leaf tissue and corresponded to 10 copies of the target molecule. The assay was specific and did not cross-react with other common cucurbit viruses found in Florida and Georgia. The performance of the exo RT-RPA was evaluated using crude extract from 21 cucurbit field samples and demonstrated that the exo RT-RPA is a rapid procedure, thus providing a promising novel alternative approach for the detection of CYSDV.

An Improved Crop Scouting Technique Incorporating Unmanned Aerial Vehicle-Assisted Multispectral Crop Imaging into Conventional Scouting Practice for Gummy Stem Blight in Watermelon.

Multispectral imaging is increasingly used in specialty crops, but its benefits in assessment of disease severity and improvements in conventional scouting practice are unknown. Multispectral imaging was conducted using an unmanned aerial vehicle (UAV), and data were analyzed for five flights from Florida and Georgia commercial watermelon fields in 2017. The fields were rated for disease incidence and severity by extension agents and plant pathologists at randomized locations (i.e., conventional scouting) followed by ratings at locations that were identified by differences in normalized difference vegetation index (NDVI) and stress index (i.e., UAV-assisted scouting). Diseases identified by the scouts included gummy stem blight, anthracnose, Fusarium wilt, Phytophthora fruit rot, Alternaria leaf spot, and cucurbit leaf crumple disease. Disease incidence and severity ratings were significantly different between conventional and UAV-assisted scouting (P < 0.01, Bhapkar/exact test). Higher severity ratings of 4 and 5 on a scale of 1 to 5 from no disease to complete loss of the canopy were more consistent after the scouts used the multispectral images in determining sampling locations. The UAV-assisted scouting locations had significantly lower green, red, and red edge NDVI values and higher stress index values than the conventional scouting areas (P < 0.05, ANOVA/Tukey), and this corresponded to areas with higher disease severity. Conventional scouting involving human evaluation remains necessary for disease validation. Multispectral imagery improved watermelon field scouting owing to increased ability to identify disease foci and areas of concern more rapidly than conventional scouting practices with early detection of diseases 20% more often using UAV-assisted scouting.

Artificial forisomes are ideal models of forisome assembly and activity that allow the development of technical devices.

Forisomes are protein polymers found in leguminous plants that have the remarkable ability to undergo reversible "muscle-like" contractions in the presence of divalent cations and in extreme pH environments. To gain insight into the molecular basis of forisome structure and assembly, we used confocal laser scanning microscopy to monitor the assembly of fluorescence-labeled artificial forisomes in real time, revealing two distinct assembly processes involving either fiber elongation or fiber alignment. We also used scanning and transmission electron microscopy and X-ray diffraction to investigate the ultrastructure of forisomes, finding that individual fibers are arranged into compact fibril bundles that disentangle with minimal residual order in the presence of calcium ions. To demonstrate the potential applications of artificial forisomes, we created hybrid protein bodies from forisome subunits fused to the B-domain of staphylococcal protein A. This allowed the functionalization of the artificial forisomes with antibodies that were then used to target forisomes to specific regions on a substrate, providing a straightforward approach to develop forisome-based technical devices with precise configurations. The functional contractile properties of forisomes are also better preserved when they are immobilized via affinity reagents rather than by direct contact to the substrate. Artificial forisomes produced in plants and yeast therefore provide an ideal model for the investigation of forisome structure and assembly and for the design and testing of tailored artificial forisomes for technical applications.

Genetic Composition of Phytophthora infestans in Canada Reveals Migration and Increased Diversity.

A dramatic increase in the incidence of late blight and changes within populations of Phytophthora infestans have been observed in various regions of Canada. In this study, the occurrence of several new genotypes of the pathogen was documented with associated phenotypes that dominated pathogen populations. Genotype US-23, previously detected only among isolates from the United States, dominated in the western Canadian provinces of British Columbia, Alberta (AB), Saskatchewan, and Manitoba (MB). Although isolates of US-23 infect both potato and tomato, these isolates were the only genotype recovered from commercial garden centers in Canada. Isolates of genotype US-8, previously dominant throughout Canada, represented the only genotype detected from the eastern Canadian provinces of New Brunswick and Prince Edward Island. Isolates of other genotypes detected in Canada included US-11 in AB, US-24 in MB, and US-22 in Ontario (ON). An additional genotype was detected in ON which appears to be a derivative of US-22 that may have arisen through sexual reproduction. However, evidence of clonal reproduction dominated among the isolates collected, and opportunities for sexual reproduction were probably limited because of a surprising geographic separation of the A1 and A2 mating types in Canada. Sensitivity of the US-22, US-23, and US-24 isolates to the fungicide metalaxyl, movement of potato seed and transplants, and weather conditions may have contributed to reduced opportunities for contact between the mating types in fields in Canada. All P. infestans isolates were readily distinguished from other related oomycetes with RG57 restriction fragment length polymorphism analysis. Long-distance movement in seed tubers and garden center transplants may have contributed to the rapid spread of the P. infestans genotypes across Canada. Tracking pathogen movement and population composition should improve the ability to predict the genotypes expected each year in different regions of Canada.

Amplification of cell signaling and disease resistance by an immunity receptor Ve1Ve2 heterocomplex in plants.

Immunity cell-surface receptors Ve1 and Ve2 protect against fungi of the genus Verticillium causing early dying, a worldwide disease in many crops. Characterization of microbe-associated molecular pattern immunity receptors has advanced our understanding of disease resistance but signal amplification remains elusive. Here, we report that transgenic plants expressing Ve1 and Ve2 together, reduced pathogen titres by a further 90% compared to plants expressing only Ve1 or Ve2. Confocal and immunoprecipitation confirm that the two receptors associate to form heteromeric complexes in the absence of the ligand and positively regulate signaling. Bioassays show that the Ve1Ve2 complex activates race-specific amplified immunity to the pathogen through a rapid burst of reactive oxygen species (ROS). These results indicate a mechanism by which the composition of a cell-surface receptor heterocomplex may be optimized to increase immunity against devastating plant diseases.

Tobacco mosaic virus infection results in an increase in recombination frequency and resistance to viral, bacterial, and fungal pathogens in the progeny of infected tobacco plants.

Our previous experiments showed that infection of tobacco (Nicotiana tabacum) plants with Tobacco mosaic virus (TMV) leads to an increase in homologous recombination frequency (HRF). The progeny of infected plants also had an increased rate of rearrangements in resistance gene-like loci. Here, we report that tobacco plants infected with TMV exhibited an increase in HRF in two consecutive generations. Analysis of global genome methylation showed the hypermethylated genome in both generations of plants, whereas analysis of methylation via 5-methyl cytosine antibodies demonstrated both hypomethylation and hypermethylation. Analysis of the response of the progeny of infected plants to TMV, Pseudomonas syringae, or Phytophthora nicotianae revealed a significant delay in symptom development. Infection of these plants with TMV or P. syringae showed higher levels of induction of PATHOGENESIS-RELATED GENE1 gene expression and higher levels of callose deposition. Our experiments suggest that viral infection triggers specific changes in progeny that promote higher levels of HRF at the transgene and higher resistance to stress as compared with the progeny of unstressed plants. However, data reported in these studies do not establish evidence of a link between recombination frequency and stress resistance.

The Luteovirus P4 Movement Protein Is a Suppressor of Systemic RNA Silencing.

The plant viral family Luteoviridae is divided into three genera: Luteovirus, Polerovirus and Enamovirus. Without assistance from another virus, members of the family are confined to the cells of the host plant's vascular system. The first open reading frame (ORF) of poleroviruses and enamoviruses encodes P0 proteins which act as silencing suppressor proteins (VSRs) against the plant's viral defense-mediating RNA silencing machinery. Luteoviruses, such as barley yellow dwarf virus-PAV (BYDV-PAV), however, have no P0 to carry out the VSR role, so we investigated whether other proteins or RNAs encoded by BYDV-PAV confer protection against the plant's silencing machinery. Deep-sequencing of small RNAs from plants infected with BYDV-PAV revealed that the virus is subjected to RNA silencing in the phloem tissues and there was no evidence of protection afforded by a possible decoy effect of the highly abundant subgenomic RNA3. However, analysis of VSR activity among the BYDV-PAV ORFs revealed systemic silencing suppression by the P4 movement protein, and a similar, but weaker, activity by P6. The closely related BYDV-PAS P4, but not the polerovirus potato leafroll virus P4, also displayed systemic VSR activity. Both luteovirus and the polerovirus P4 proteins also showed transient, weak local silencing suppression. This suggests that systemic silencing suppression is the principal mechanism by which the luteoviruses BYDV-PAV and BYDV-PAS minimize the effects of the plant's anti-viral defense.

Complete Genome Sequence of Phytopathogenic Pectobacterium atrosepticum Bacteriophage Peat1.

Pectobacterium atrosepticum is a common phytopathogen causing significant economic losses worldwide. To develop a biocontrol strategy for this blackleg pathogen of solanaceous plants, P. atrosepticum bacteriophage Peat1 was isolated and its genome completely sequenced. Interestingly, morphological and sequence analyses of the 45,633-bp genome revealed that phage Peat1 is a member of the family Podoviridae and most closely resembles the Klebsiella pneumoniae bacteriophage KP34. This is the first published complete genome sequence of a phytopathogenic P. atrosepticum bacteriophage, and details provide important information for the development of biocontrol by advancing our understanding of phage-phytopathogen interactions.

Priming with a double-stranded DNA virus alters Brassica rapa seed architecture and facilitates a defense response.

BACKGROUND: Abiotic and biotic stresses alter genome stability and physiology of plants. Under some stressful situations, a state of stress tolerance can be passed on to the offspring rendering them more suitable to stressful events than their parents. In plants, the exploration of transgenerational response has remained exclusive to model species, such as Arabidopsis thaliana. Here, we expand transgenerational research to include Brassica rapa, a close relative to economically important plant canola (Brassica napus), as it is exposed to the biotic stress of a double-stranded DNA virus Cauliflower mosaic virus (CaMV). RESULTS: Parent plants exposed to a low dose of 50ng purified CaMV virions just prior to the bolting stage produced significantly larger seeds than mock inoculated and healthy treatments. The progeny from these large seeds displayed resistance to the pathogen stress applied in the parental generation. Differences in defense pathways involving fatty acids, and primary and secondary metabolites were detected by de novo transcriptome sequencing of CaMV challenged progeny exhibiting different levels of resistance. CONCLUSIONS: Our study highlights biological and cellular processes that may be linked to the growth and yield of economically important B. rapa, in a transgenerational manner. Although much remains unknown as to the mechanisms behind transgenerational inheritance, our work shows a disease resistance response that persists for several weeks and is associated with an increase in seed size. Evidence suggests that a number of changes involved in the persistent stress adaption are reflected in the transcriptome. The results from this study demonstrate that treating B. rapa with dsDNA virus within a critical time frame and with a specified amount of infectious pathogen produces economically important agricultural plants with superior coping strategies for growing in unfavorable conditions.

Small RNA sequencing of Potato leafroll virus-infected plants reveals an additional subgenomic RNA encoding a sequence-specific RNA-binding protein.

Potato leafroll virus (PLRV) is a positive-strand RNA virus that generates subgenomic RNAs (sgRNA) for expression of 3' proximal genes. Small RNA (sRNA) sequencing and mapping of the PLRV-derived sRNAs revealed coverage of the entire viral genome with the exception of four distinctive gaps. Remarkably, these gaps mapped to areas of PLRV genome with extensive secondary structures, such as the internal ribosome entry site and 5' transcriptional start site of sgRNA1 and sgRNA2. The last gap mapped to ∼500 nt from the 3' terminus of PLRV genome and suggested the possible presence of an additional sgRNA for PLRV. Quantitative real-time PCR and northern blot analysis confirmed the expression of sgRNA3 and subsequent analyses placed its 5' transcriptional start site at position 5347 of PLRV genome. A regulatory role is proposed for the PLRV sgRNA3 as it encodes for an RNA-binding protein with specificity to the 5' of PLRV genomic RNA.

Complete genomic sequence of a Rubus yellow net virus isolate and detection of genome-wide pararetrovirus-derived small RNAs.

Rubus yellow net virus (RYNV) was cloned and sequenced from a red raspberry (Rubus idaeus L.) plant exhibiting symptoms of mosaic and mottling in the leaves. Its genomic sequence indicates that it is a distinct member of the genus Badnavirus, with 7932bp and seven ORFs, the first three corresponding in size and location to the ORFs found in the type member Commelina yellow mottle virus. Bioinformatic analysis of the genomic sequence detected several features including nucleic acid binding motifs, multiple zinc finger-like sequences and domains associated with cellular signaling. Subsequent sequencing of the small RNAs (sRNAs) from RYNV-infected R. idaeus leaf tissue was used to determine any RYNV sequences targeted by RNA silencing and identified abundant virus-derived small RNAs (vsRNAs). The majority of the vsRNAs were 22-nt in length. We observed a highly uneven genome-wide distribution of vsRNAs with strong clustering to small defined regions distributed over both strands of the RYNV genome. Together, our data show that sequences of the aphid-transmitted pararetrovirus RYNV are targeted in red raspberry by the interfering RNA pathway, a predominant antiviral defense mechanism in plants.