Plant Extracellular Vesicles: Current Landscape and Future Directions.

Plant cells secrete membrane-enclosed micrometer- and nanometer-sized vesicles that, similarly to the extracellular vesicles (EVs) released by mammalian or bacterial cells, carry a complex molecular cargo of proteins, nucleic acids, lipids, and primary and secondary metabolites. While it is technically complicated to isolate EVs from whole plants or their tissues, in vitro plant cell cultures provide excellent model systems for their study. Plant EVs have been isolated from the conditioned culture media of plant cell, pollen, hairy root, and protoplast cultures, and recent studies have gathered important structural and biological data that provide a framework to decipher their physiological roles and unveil previously unacknowledged links to their diverse biological functions. The primary function of plant EVs seems to be in the secretion that underlies cell growth and morphogenesis, cell wall composition, and cell-cell communication processes. Besides their physiological functions, plant EVs may participate in defence mechanisms against different plant pathogens, including fungi, viruses, and bacteria. Whereas edible and medicinal-plant-derived nanovesicles isolated from homogenised plant materials ex vivo are widely studied and exploited, today, plant EV research is still in its infancy. This review, for the first time, highlights the different in vitro sources that have been used to isolate plant EVs, together with the structural and biological studies that investigate the molecular cargo, and pinpoints the possible role of plant EVs as mediators in plant-pathogen interactions, which may contribute to opening up new scenarios for agricultural applications, biotechnology, and innovative strategies for plant disease management.

Nanopore Technology Applied to Targeted Detection of Tomato Brown Rugose Fruit Virus Allows Sequencing of Related Viruses and the Diagnosis of Mixed Infections.

Tomato (Solanum lycopersicum) plants from a commercial glasshouse were identified with symptoms compatible with a tomato brown rugose fruit virus (ToBRFV) infection. Reverse transcription-PCR and quantitative PCR confirmed the presence of ToBRFV. Subsequently, the same RNA sample and a second from tomato plants infected with a similar tobamovirus, tomato mottle mosaic virus (ToMMV), were extracted and processed for high-throughput sequencing with the Oxford Nanopore Technology (ONT). For the targeted detection of ToBRFV, the two libraries were synthesized by using six ToBRFV sequence-specific primers in the reverse transcription step. This innovative target enrichment technology enabled deep coverage sequencing of ToBRFV, with 30% of the total reads mapping to the target virus genome and 57% mapping to the host genome. The same set of primers applied to the ToMMV library generated 5% of the total reads mapping to the latter virus, indicating that sequencing of similar, non-target viral sequences was also allowed. Further, the complete genome of pepino mosaic virus (PepMV) was also sequenced from the ToBRFV library, thus suggesting that, even using multiple sequence-specific primers, a low rate of off-target sequencing can usefully provide additional information on unexpected viral species coinfecting the same samples in an individual assay. These results demonstrate that targeted nanopore sequencing can specifically identify viral agents and has sufficient sensitivity towards non-target organisms to provide evidence of mixed virus infections.

Recombinant Expression of Archaeal Superoxide Dismutases in Plant Cell Cultures: A Sustainable Solution with Potential Application in the Food Industry.

Superoxide dismutase (SOD) is a fundamental antioxidant enzyme that neutralises superoxide ions, one of the main reactive oxygen species (ROS). Extremophile organisms possess enzymes that offer high stability and catalytic performances under a wide range of conditions, thus representing an exceptional source of biocatalysts useful for industrial processes. In this study, SODs from the thermo-halophilic Aeropyrum pernix (SODAp) and the thermo-acidophilic Saccharolobus solfataricus (SODSs) were heterologously expressed in transgenic tomato cell cultures. Cell extracts enriched with SODAp and SODSs showed a remarkable resistance to salt and low pHs, respectively, together with optimal activity at high temperatures. Moreover, the treatment of tuna fillets with SODAp-extracts induced an extension of the shelf-life of this product without resorting to the use of illicit substances. The results suggested that the recombinant plant extracts enriched with the extremozymes could find potential applications as dietary supplements in the nutrition sector or as additives in the food preservation area, representing a more natural and appealing alternative to chemical preservatives for the market.

Potato Virus Y Infection Alters Small RNA Metabolism and Immune Response in Tomato.

Potato virus Y (PVY) isolate PVYC-to induces growth reduction and foliar symptoms in tomato, but new vegetation displays symptom recovery at a later stage. In order to investigate the role of micro(mi)RNA and secondary small(s)RNA-regulated mechanisms in tomato defenses against PVY, we performed sRNA sequencing from healthy and PVYC-to infected tomato plants at 21 and 30 days post-inoculation (dpi). A total of 792 miRNA sequences were obtained, among which were 123 canonical miRNA sequences, many isomiR variants, and 30 novel miRNAs. MiRNAs were mostly overexpressed in infected vs. healthy plants, whereas only a few miRNAs were underexpressed. Increased accumulation of isomiRs was correlated with viral infection. Among miRNA targets, enriched functional categories included resistance (R) gene families, transcription and hormone factors, and RNA silencing genes. Several 22-nt miRNAs were shown to target R genes and trigger the production of 21-nt phased sRNAs (phasiRNAs). Next, 500 phasiRNA-generating loci were identified, and were shown to be mostly active in PVY-infected tissues and at 21 dpi. These data demonstrate that sRNA-regulated host responses, encompassing miRNA alteration, diversification within miRNA families, and phasiRNA accumulation, regulate R and disease-responsive genes. The dynamic regulation of miRNAs and secondary sRNAs over time suggests a functional role of sRNA-mediated defenses in the recovery phenotype.

Ozone treatments activate defence responses against Meloidogyne incognita and Tomato spotted wilt virus in tomato.

BACKGROUND: Ozonated water (O3 wat) soil drench and/or foliar spray applications were evaluated for their potential to control the root-knot nematode Meloidogyne incognita (RKN) and the airborne pathogen Tomato spotted wilt virus (TSWV) in tomato. We investigated how O3 wat modulates the salicylic acid/jasmonic acid/ethylene (SA/JA/ET) signalling network in the host, locally and systemically, to induce resistance to nematode and virus. RESULTS: The application as soil drench was effective in reducing the number of galls and egg masses, but did not reduce the incidence and severity of TSWV infection. Conversely, O3 wat applied by foliar spray decreased TSWV disease incidence and severity (-20%), but was not able to control M. incognita infection. SA-related genes were generally upregulated in both locally treated and systemically reached tissues, showing a positive action of the O3 wat treatment on SA signalling. Neither O3 wat application method significantly altered JA-related gene expression in either direction. ET-related genes were differentially regulated by root or leaf treatments, indicating that O3 wat may have different effects on ET-mediated signalling in different organs. JA/ET/SA related pathways were differentially modulated by O3 wat in the presence of either RKN or TSWV. CONCLUSION: O3 wat had a higher efficacy when applied directly to organs challenged by the pathogens, although it was potentially able to stimulate defence responses through the activation of SA signalling. Owing to its safety and effectiveness in controlling nematode and virus infections, O3 wat can be considered as a possible alternative tool for sustainable disease management practices. © 2019 Society of Chemical Industry.

Rapid identification of tomato Sw-5 resistance-breaking isolates of Tomato spotted wilt virus using high resolution melting and TaqMan SNP Genotyping assays as allelic discrimination techniques.

In tomato, resistance to Tomato spotted wilt virus (TSWV) is conferred by the dominant gene, designated Sw-5. Virulent Sw-5 resistance breaking (SRB) mutants of TSWV have been reported on Sw-5 tomato cultivars. Two different PCR-based allelic discrimination techniques, namely Custom TaqMan™ SNP Genotyping and high-resolution melting (HRM) assays, were developed and compared for their ability to distinguish between avirulent (Sw-5 non-infecting, SNI) and SRB biotypes. TaqMan assays proved to be more sensitive (threshold of detection in a range of 50-70 TSWV RNA copies) and more reliable than HRM, assigning 25 TSWV isolates to their correct genotype with an accuracy of 100%. Moreover, the TaqMan SNP assays were further improved developing a rapid and simple protocol that included crude leaf extraction for RNA template preparations. On the other hand, HRM assays showed higher levels of sensitivity than TaqMan when used to co-detect both biotypes in different artificial mixtures. These diagnostic assays contributed to gain preliminary information on the epidemiology of TSWV isolates in open field conditions. In fact, the presented data suggest that SRB isolates are present as stable populations established year round, persisting on both winter (globe artichoke) and summer (tomato) crops, in the same cultivated areas of Southern Italy.

Prosystemin overexpression induces transcriptional modifications of defense-related and receptor-like kinase genes and reduces the susceptibility to Cucumber mosaic virus and its satellite RNAs in transgenic tomato plants.

Systemin is a plant signal peptide hormone involved in the responses to wounding and insect damage in the Solanaceae family. It works in the same signaling pathway of jasmonic acid (JA) and enhances the expression of proteinase inhibitors. With the aim of studying a role for systemin in plant antiviral responses, a tomato (Solanum lycopersicum) transgenic line overexpressing the prosystemin cDNA, i.e. the systemin precursor, was inoculated with Cucumber mosaic virus (CMV) strain Fny supporting either a necrogenic or a non-necrogenic satellite RNA (satRNA) variant. Transgenic plants showed reduced susceptibility to both CMV/satRNA combinations. While symptoms of the non-necrogenic inoculum were completely suppressed, a delayed onset of lethal disease occurred in about half of plants challenged with the necrogenic inoculum. RT-qPCR analysis showed a correlation between the systemin-mediated reduced susceptibility and the JA biosynthetic and signaling pathways (e.g. transcriptional alteration of lipoxygenase D and proteinase inhibitor II). Moreover, transgenically overexpressed systemin modulated the expression of a selected set of receptor-like protein kinase (RLK) genes, including some playing a known role in plant innate immunity. A significant correlation was found between the expression profiles of some RLKs and the systemin-mediated reduced susceptibility to CMV/satRNA. These results show that systemin can increase plant defenses against CMV/satRNA through transcriptional reprogramming of diverse signaling pathways.

Transgenic resistance.

Transgenic resistance to plant viruses is an important technology for control of plant virus infection, which has been demonstrated for many model systems, as well as for the most important plant viruses, in terms of the costs of crop losses to disease, and also for many other plant viruses infecting various fruits and vegetables. Different approaches have been used over the last 28 years to confer resistance, to ascertain whether particular genes or RNAs are more efficient at generating resistance, and to take advantage of advances in the biology of RNA interference to generate more efficient and environmentally safer, novel "resistance genes." The approaches used have been based on expression of various viral proteins (mostly capsid protein but also replicase proteins, movement proteins, and to a much lesser extent, other viral proteins), RNAs [sense RNAs (translatable or not), antisense RNAs, satellite RNAs, defective-interfering RNAs, hairpin RNAs, and artificial microRNAs], nonviral genes (nucleases, antiviral inhibitors, and plantibodies), and host-derived resistance genes (dominant resistance genes and recessive resistance genes), and various factors involved in host defense responses. This review examines the above range of approaches used, the viruses that were tested, and the host species that have been examined for resistance, in many cases describing differences in results that were obtained for various systems developed in the last 20 years. We hope this compilation of experiences will aid those who are seeking to use this technology to provide resistance in yet other crops, where nature has not provided such.

Pea powdery mildew er1 resistance is associated to loss-of-function mutations at a MLO homologous locus.

The powdery mildew disease affects several crop species and is also one of the major threats for pea (Pisum sativum L.) cultivation all over the world. The recessive gene er1, first described over 60 years ago, is well known in pea breeding, as it still maintains its efficiency as a powdery mildew resistance source. Genetic and phytopathological features of er1 resistance are similar to those of barley, Arabidopsis, and tomato mlo powdery mildew resistance, which is caused by the loss of function of specific members of the MLO gene family. Here, we describe the obtainment of a novel er1 resistant line by experimental mutagenesis with the alkylating agent diethyl sulfate. This line was found to carry a single nucleotide polymorphism in the PsMLO1 gene sequence, predicted to result in premature termination of translation and a non-functional protein. A cleaved amplified polymorphic sequence (CAPS) marker was developed on the mutation site and shown to be fully co-segregating with resistance in F(2) individuals. Sequencing of PsMLO1 from three powdery mildew resistant cultivars also revealed the presence of loss-of-function mutations. Taken together, results reported in this study strongly indicate the identity between er1 and mlo resistances and are expected to be of great breeding importance for the development of resistant cultivars via marker-assisted selection.

Characterization of the interactions between Cucumber mosaic virus and Potato virus Y in mixed infections in tomato.

Mixed infection with the SON41 strain of Potato virus Y (PVY-SON41) in tomato increased accumulation of RNAs of strains Fny and LS of Cucumber mosaic virus (CMV-Fny and CMV-LS, respectively) and enhanced disease symptoms. By contrast, replication of PVY-SON41 was downregulated by CMV-Fny and this was due to the CMV-Fny 2b protein. The CMV-FnyΔ2b mutant was unable to systemically invade the tomato plant because its movement was blocked at the bundle sheath of the phloem. The function needed for invading the phloem was complemented by PVY-SON41 in plants grown at 22°C whereas this complementation was not necessary in plants grown at 15°C. Mutations in the 2b protein coding sequence of CMV-Fny as well as inhibition of translation of the 2a/2b overlapping region of the 2a protein lessened both the accumulation of viral RNAs and the severity of symptoms. Both of these functions were complemented by PVY-SON41. Infection of CMV-Fny supporting replication of the Tfn-satellite RNA reduced the accumulation of CMV RNA and suppressed symptom expression also in plants mixed-infected with PVY-SON41. The interaction between CMV and PVY-SON41 in tomato exhibited different features from that documented in other hosts. The results of this work are relevant from an ecological and epidemiological perspective due to the frequency of natural mixed infection of CMV and PVY in tomato.

Evaluation of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in infected tomato plants.

The quantification of messenger RNA expression levels by real-time reverse-transcription polymerase chain reaction requires the availability of reference genes that are stably expressed regardless of the experimental conditions under study. We examined the expression variations of a set of eight candidate reference genes in tomato leaf and root tissues subjected to the infection of five taxonomically and molecularly different plant viruses and a viroid, inducing diverse pathogenic effects on inoculated plants. Parallel analyses by three commonly used dedicated algorithms, geNorm, NormFinder and BestKeeper, showed that different viral infections and tissues of origin influenced, to some extent, the expression levels of these genes. However, all algorithms showed high levels of stability for glyceraldehyde 3-phosphate dehydrogenase and ubiquitin, indicated as the most suitable endogenous transcripts for normalization in both tissue types. Actin and uridylate kinase were also stably expressed throughout the infected tissues, whereas cyclophilin showed tissue-specific expression stability only in root samples. By contrast, two widely employed reference genes, 18S ribosomal RNA and elongation factor 1α, demonstrated highly variable expression levels that should discourage their use for normalization. In addition, expression level analysis of ascorbate peroxidase and superoxide dismutase showed the modulation of the two genes in virus-infected tomato leaves and roots. The relative quantification of the two genes varied according to the reference genes selected, thus highlighting the importance of the choice of the correct normalization method in such experiments.

RNAi-mediated viral immunity requires amplification of virus-derived siRNAs in Arabidopsis thaliana.

In diverse eukaryotic organisms, Dicer-processed, virus-derived small interfering RNAs direct antiviral immunity by RNA silencing or RNA interference. Here we show that in addition to core dicing and slicing components of RNAi, the RNAi-mediated viral immunity in Arabidopsis thaliana requires host RNA-directed RNA polymerase (RDR) 1 or RDR6 to produce viral secondary siRNAs following viral RNA replication-triggered biogenesis of primary siRNAs. We found that the two antiviral RDRs exhibited specificity in targeting the tripartite positive-strand RNA genome of cucumber mosaic virus (CMV). RDR1 preferentially amplified the 5'-terminal siRNAs of each of the three viral genomic RNAs, whereas an increased production of siRNAs targeting the 3' half of RNA3 detected in rdr1 mutant plants appeared to be RDR6-dependent. However, siRNAs derived from a single-stranded 336-nucleotide satellite RNA of CMV were not amplified by either antiviral RDR, suggesting avoidance of the potent RDR-dependent silencing as a strategy for the molecular parasite of CMV to achieve preferential replication. Our work thus identifies a distinct mechanism for the amplification of immunity effectors, which together with the requirement for the biogenesis of endogenous siRNAs, may play a role in the emergence and expansion of eukaryotic RDRs.

Differential effects of mild and severe Cucumber mosaic virus strains in the perturbation of MicroRNA-regulated gene expression in tomato map to the 3' sequence of RNA 2.

Viral infections interfere with the microRNA (miRNA)-mediated regulation of gene expression, determining developmental defects. In tomato leaves, the accumulation levels of six miRNA species and their target transcripts corresponding to transcription factors with roles in plant development and leaf morphogenesis and two genes involved in the short RNA processing, DCL1 and AGO1, were significantly enhanced upon infection with the severe strain Cucumber mosaic virus (CMV)-Fny, while that of AGO4 was reduced. In plants harboring the infection of the mild strain CMV-LS, the effects on miRNA pathway were reduced, although AGO1, DCL1, and NAC1 also were shown to overaccumulate during infections exhibiting a mild phenotype. The use of the recombinant strain CMV-Fny(LS2b), in which the 3'-terminal region of CMV-Fny RNA 2, including the 2b coding sequence, was replaced with the corresponding region of CMV-LS RNA 2, provided evidence that the exchanged region was implicated in the perturbation of miRNA metabolism. In tomato plants infected with CMV-Fny supporting the ameliorative satellite (sat)RNA variant Tfn-satRNA, the symptomless phenotype correlated, with the exception of NAC1 upregulation, with the absence of effects on mitochondrial RNA and miRNA expression. Some of the aspects of miRNA pathway perturbation described were peculiar to CMV-tomato interactions and involved in the etiology of the disease phenotype elicited in this host.

Analysis of mechanisms involved in the Cucumber mosaic virus satellite RNA-mediated transgenic resistance in tomato plants.

Transgenic tomato (Lycopersicon esculentum Mill. cv. UC82) plants expressing a benign variant of Cucumber mosaic virus satellite RNA (CMV Tfn-satRNA) were generated. The transformed plants did not produce symptoms when challenged with a satRNA-free strain of CMV (CMV-FL). The same plant lines initially were susceptible to necrosis elicited by a CMV strain supporting a necrogenic variant of satRNA (CMV-77), but a phenotype of total recovery from the necrosis was observed in the newly developing leaves. The features of the observed resistance were analyzed and are consistent with two different mechanisms of resistance. In transgenic plants inoculated with CMV-FL strain, the symptomless phenotype was correlated to the down-regulation of CMV by Tfn-satRNA, amplified from the transgene transcripts, as the first resistance mechanism. On the other hand, the delayed resistance to CMV-77 in transgenic tomato lines was mediated by a degradation process that targets satRNAs in a sequence-specific manner. Evidence is provided for a correlation between a reduced accumulation level of transgenic messenger Tfn-satRNA, the accumulation of small (approximately 23 nucleotides) RNAs with sequence homology to satRNAs, the progressively reduced accumulation of 77-satRNA in infected tissues, and the transition in infected plants from diseased to healthy. Thus, events leading to the degradation of satRNA sequences indicate a role for RNA silencing as the second mechanism determining resistance of transgenic tomato lines.

Generation of siRNAs by T-DNA sequences does not require active transcription or homology to sequences in the plant.

Delivery into plants of T-DNAs containing promoter, terminator, or coding sequences generated small interfering RNAs (siRNAs) specific to each type of sequence. When both promoter and transcribed sequences were simultaneously present in the T-DNA, accumulation of siRNAs to transcribed sequences was favored over accumulation of siRNAs to the nontranscribed upstream promoter sequences. The generation of specific siRNA sequences occurred even in the absence of T-DNA homology to sequences in the plant. Delivery of T-DNA, with homology to the transgene limited to the nontranscribed cauliflower mosaic virus 35S promoter (35SP) and the transcribed nopaline synthase transcription termination (NosT)signal sequences, into transgenic plants expressing the green fluorescent protein (GFP), generated siRNAs in infiltrated tissues to both 35SP (35SsiRNAs) and NosT (NosTsiRNAs), but not to the GFP sequence (GFPsiRNAs). In infiltrated tissues, the 35SsiRNAs failed to trigger the transcriptional silencing of the transgene, accumulation of 35SsiRNAs could be prevented by the potyviral HC-Pro, and the NosTsiRNAs required an initial amplification to trigger efficient transgene silencing, which is mediated by transcripts from the exogenous T-DNA, and not from the transgene. In upper leaves, silencing correlated with the presence of GFPsiRNAs and the absence of 35SsiRNAs, confirming that its spread was posttranscriptionally mediated by the transgene mRNA.

In situ localization and tissue distribution of the replication-associated proteins of Cucumber mosaic virus in tobacco and cucumber.

The replication-associated proteins encoded by Cucumber mosaic virus (CMV), the 1a and 2a proteins, were detected by immunogold labeling in two host species of this virus, tobacco (Nicotiana tabacum) and cucumber (Cucumis sativus). In both hosts, the 1a and 2a proteins colocalized predominantly to the vacuolar membranes, the tonoplast. While plus-strand CMV RNAs were found distributed throughout the cytoplasm by in situ hybridization, minus-strand CMV RNAs were barely detectable but were found associated with the tonoplast. In both cucumber and tobacco, 2a protein was detected at higher densities than 1a protein. The 1a and 2a proteins also showed quantitative differences with regard to tissue distributions in tobacco and cucumber. About three times as much 2a protein was detected in CMV-infected cucumber tissues as in CMV-infected tobacco tissues. In tobacco, high densities of these proteins were observed only in vascular bundle cells of minor veins. In contrast, in cucumber, high densities of 1a and 2a proteins were observed in mesophyll cells, followed by epidermis cells, with only low levels being observed in vascular bundle cells. Differences were also observed in the distributions of 2a protein and capsid protein in vascular bundle cells of the two host species. These observations may represent differences in the relative rates of tissue infection in different hosts or differences in the extent of virus replication in vascular tissues of different hosts.