Molecular tools to investigate Sharka disease in Prunus species

C H A P T E R

10 Molecular tools to investigate Sharka disease in Prunus species Gloria De Moria, Federica Savazzinib, Filippo Geunac a

Università degli Studi di Udine, Department of Agricultural, Food, Environmental and Animal Sciences, Udine, Italy bUniversità degli Studi di Bologna, Department of Agricultural and Food Sciences DISTAL, Bologna, Italy cUniversità degli Studi di Milano, Department of Agricultural and Environmental Sciences—Production, Landscapes, Agroenergy, Milano, Italy

10.1  The Sharka disease Sharka is a quarantine pest considered one of the most devastating virus diseases of tree plant species of agronomic interest. Various strains of its causal agent, the Plum pox virus (PPV), are capable of affecting peach, nectarine, apricot, plum, almond, sweet and sour cherry, as well as various other Prunus and non-Prunus species. The literature on Sharka dates back to the beginning of the 20th century when Atanassov (1932) described the occurrence of what he called “Plum Pox” as a new virus disease. Since then, several reports appeared in the literature concerning its discovery in various stone fruit species and geographic areas (as shown in Fig. 10.1, adapted from Rimbaud et al., 2015). As of December 2018, a bibliographical search of the PubMed database (Canese and Weis, 2013) with the query ‘(Plum pox virus) OR (Sharka) OR (potyvirus)’ returned 2371 occurrences of publications relating to peer-reviewed research. The three main subdomains of investigation pertaining this subject are: (a) the diagnostics of virus presence in plant and (b) the genotyping of plant resistance or tolerance to infection and (c) the physiological aspects of plant-virus interaction in both Prunus and model grass species. All these aspects are reviewed in this section. Sharka first appearance in Europe dates back to the first decades of the 20th century. Since then, the disease has spread progressively to most European areas, around the Mediterranean basin and the Near and Middle East. It has also spread to South and North America and Asia (Barba et al., 2011), with the exception of Australia, New Zealand, South Africa. During the last decades, Sharka disease has had a significant agronomic impact and resulted in major economic losses. Several studies reported that the cost of Sharka management is enormous. The costs of the disease management were already esteemed to exceed 10 billion euros back in 2006 (Cambra et al., 2006).

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10.  Molecular tools to investigate Sharka disease in Prunus species

FIG. 10.1  World map distribution of PPV. Black areas, PPV present; grey areas, no data on PPV occurrence; light grey areas, PPV absent; white areas, no Prunus crops. Modified from: Center for Agriculture and Bioscience International CABI website (www.cabi.org).

Sharka affected cultivars can reach, in some cases, even 100% losses (Cambra et al., 2006; García et al., 2014). Such economic losses, as a result of extensive plant infections, are not only due to the impact of decreased fruit quality but include high costs associated to diagnostics, prevention strategies and containment of the disease by means of quarantine and eradication (García et al., 2014). Europe particularly suffers the highest economic impact although European countries differ as for the level of disease diffusion and the incidence of various virus strains (Sihelská et al., 2017 and references therein). Trading of infected plant vegetative material (rootstocks) is the common way by which the virus is diffused at long distance. In nature, the virus, can be efficiently transmitted by aphid vectors in a non persistent mode. Insecticide treatment in orchards to reduce the aphid vector pressure does not prove effective against non-persistent viruses such as PPV. Moreover, as Sharka symptoms are highly dependent on both environmental conditions (typically vegetative stage of the trees and temperature) and the sensitivity of the host plant, disease control is very difficult (Clemente-Moreno et al., 2015). Typically, sharka symptoms feature chlorotic rings or spots, leaf vein clearing and distortion, necrotic areas and deformation on fruits, as shown in Fig. 10.2 (Sochor et al., 2012). An additional aspect of the PPV infection is the



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FIG. 10.2  Symptoms of the plum pox virus on stone fruits flowers and leaves.

decline of the fruit quality, in particular when symptoms become manifest: fruits have reduced weight and sugar content (Usenik and Marn, 2017). Because of the magnitude of the damages caused by PPV and the high spread of the virus, the European and Mediterranean Plant Protection Organization (EPPO/OEPP) has included PPV in the list of quarantine pathogens and the federal government of the United States of America has classified the virus among the top ten most important adversities for its agriculture (Public Health Security and Bioterrorism Act, 2002). The etiological agent of sharka is Plum pox virus (PPV), belonging to the Potyvirus genus (García et al., 2014; Revers and García, 2015). The PPV genome consists of a single-filament, positive-sense RNA, of about 9800 nucleotides in length, encapsidated by a single coat protein (CP) to form a rod-shaped particle. The genomic RNA carries a covalently linked virusencoded protein (VPg) at the 5′ end and poly(A) tail at the 3′ one. It features a long open reading frame (ORF) that is translated into a large polyprotein precursor initiating from the second AUG codon (García et al., 2014). Three virus-encoded proteinases catalyze the cleavage of the polyprotein that generate at least ten mature protein products. Besides, another

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protein, termed P3N-PIPO, is potentially produced as the result of a translational frameshift. The limited genome coding capacity of PPV genome, like any plant virus, determines its dependance on the host machinery for its life cycle, making it an intracellular parasite. In order to accomplish its infection cycle, PPV exploits the multifunctional features of its proteins and the contribution of a set of plant factors (García et al., 2014; Revers and García, 2015). The PPV isolates described in the literature have been grouped into various monophyletic strains with specific symptomatology, host range, genome sequence and insect vector transmissibility (García et al., 2014). Strains M (Markus), D (Dideron) and Rec (Recombinant) are the most relevant and widespread. Recently, in the frame of an international project (Sharka Containment, acronym SharCo), a systematic collection of sequence data from world-wide originating PPV isolates was generated (http://www.sharco.eu/). PPV tends to accumulate mutations in different genome parts following a long-term maintenance in Prunus host plants and passage in Nicotiana benthamiana (Vozárová et al., 2013). Moreover it has been demonstrated that the virus undergoes extensive post-translational modifications (PTM). PPV virions (strain D), obtained from herbaceous hosts, are extensively modified in vivo by O-GlcNAcylation in the N-terminus of CP and by phosphorylation along the whole protein. Such PTMs are operated by Secret Agent, an O-linked N-acetylglucosamine transferase identified in Arabidopsis thaliana, and by one or more yet unidentified kinase(s) (Pérez et al., 2006, 2013; Chen et al., 2005; Fernàndez-Fernàndez et al., 2002; Scott et al., 2006; Kim et al., 2011). During infection O-GlcNAcylation of CP exerts a key role intervening in virion assembly and stability; at the same time, phosphorylation appears to modulate, alone or also in combination with O-GlcNAcylation, different CP-mediated processes during the virus cycle (Martínez-Turiño et al., 2018).

10.1.1  Physiological aspects of Sharka infection PPV infection has been reported to be the result of various interactions between host factors and virus involving the signaling and metabolic pathways controlling both diverse physiological behaviors and morphological patterns (Decroocq et al., 2006; Nováková et al., 2018). Unfortunately, the extensive research done has not been able to clarify the link between virus strain type and specific biological properties such as host range, pathogenicity, and epidemiological features (Sihelská et al., 2017). Beside the model species like Arabidopsis and N. benthamiana, the GF305 peach cultivar, showing a great susceptibility to PPV, is commonly used in PPV resistance tests on Prunus, under both in vivo (Martinez-Gomez and Dicenta, 2000) and in vitro conditions (ClementeMoreno et al., 2011; Monticelli et al., 2012; Clemente-Moreno et al., 2015). Anyway, it is important to note that, in general, these investigation methods suffer several limitations, basically due to the uneven distribution patterns of PPV in plant tissues. The recent proteomic characterization of plant response to PPV infection in N. benthamiana (Nováková et al., 2018) provides additional evidence in this regard. In this work the Authors have identified 38 unique plant proteins with a significantly altered profile following infection. Interestingly, the highest differences in profiles were identified for photosynthesis- related proteins, in particular from the Calvin-Benson cycle, whereby plants infected with the more aggressive PPV-CR strain showed the most severe changes than those with the milder



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PPV-C strain. As for the physiological response, a set of changes were detected including a marked reduction in the amount of photosynthetic pigments extracted from the leaves (in the PPV-CR strain-infected plants) and in the relative abundance of proteins that are involved in the activation of photosynthetic potential, modification of carbohydrate and amino acid metabolism. The change in the amino acid metabolism, in particular, may explain a modification of plant growth and stimulate energy formation through gluconeogenesis during PPV infection. In addition, a higher accumulation of H2O2 in infected leaves activates the glutathione synthesis, proving a crucial response in plant defense and development (Clemente-Moreno et al., 2015).

10.1.2  Oxidative stress Plant cell ultrastructure of infected plants undergoes changes induced by PPV infection that have been investigated in model grass species as well as Prunus species. PPV infection has been reported to cause an increased production of reactive oxygen species (ROS), changes of the antioxidative metabolism plus proteome changes, particularly affecting the chloroplast. Moreover, modifications in the chloroplast ultrastructure parallel the alteration of fluorescence parameters in plants following PPV infection. Among the cell ultrastructural alterations, the establishment of oxidative stress during viral disease development is correlated to the increase in the number and size of plastoglobules observed in PPV-infected plants (Hernández et al., 2004, 2006; Diaz-Vivancos et al., 2008; Clemente-Moreno et al., 2015).

10.2  Genetic resources and plant breeding 10.2.1  Mapping resistance and susceptibility factors Strategies used to control the disease dispersion include only the use of healthy plant material and the eradication of infected plants. This is the reason why the breeding programs are focused on the research of resistance sources and the development of resistance genotypes. Moreover, a genetic linkage map that includes the traits associated with Sharka resistance could be a useful instruments for the marker-assisted selection (MAS) in breeding programs. However, the development of genetic studies on Prunus species has been limited, because of the common difficulties that perennial tree crops involve, very long generation times and limited population sizes being two examples. The first Prunus Reference Map was based on the F2 progeny from Texas (almond) x Earlygold (peach) cross T x E (Joobeur et al., 1998; Aranzana et al., 2003; Dirlewanger et al., 2004; Howad et al., 2005). This reference map has allowed mapping several Prunus species like peach, plum and apricot, thanks to the strict colinearity of genomes of those species. In the European plum (P. domestica) both quantitative and qualitative (hypersensitivity) sources of resistance have been identified. The former is found for example in the ‘Stanley’, ‘President’ and ‘Ruth Gerstetter’ varieties, while the latter was first found in the cultivar ‘Jojo’. In peach, despite the extensive screening of several varieties, no sources of PPV resistance have been found. However, several cultivars show significant differences in susceptibility to

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the disease. Nine peach cultivars have been found to be tolerant to PPV, the observed tolerance being probably related to a quantitative source of resistance (Decroocq et al., 2005). A resistance character has been found also in some almond cultivars (P. dulcis), at least against the less virulent PPV-D strain. This character can be transferred to peach through interspecific hybridizations (Martínez-Gómez et al., 2004). Moreover, a kind of polygenic resistance has been reported in Prunus davidiana (Decroocq et al., 2005; Marandel et al., 2009), a species closely related to peach (P. persica (L.) Batsch.). In order to address the severe threat to the cultivations of apricot and the other species of Prunus, genetic breeding programs were established in Spain (Egea et al., 1999; Badenes et al., 2002), France (Audergon, 1995), Italy (Bassi et al., 1995) and Greece (Karayiannis et al., 1999) with the common aim to introduce PPV resistance into élite cultivars. Early studies of resistance to PPV in apricot showed that resistance appeared to be under simple genetic control involving at least one gene locus (Dicenta et  al., 2000; Karayiannis, 2008). Anyway, the comparative analysis of the different linkage maps of Prunus shows synteny between species for some QTL. Among these the genetic determinant which is able to explain a good fraction of the variability linked with PPV resistance is present in the first part of the linkage group 1 in the apricot cultivars. PPV resistance phenotyping is a lengthy procedure, in which standardization is hindered by environmental factors and the physiological state of both plant and rootstock, that affect the manifestation of the trait. Beside environmental factors, translocation of the virus and development of the infection may be affected by minor as yet unknown factors (Soriano et al., 2008). Differences between resistant cultivars in the restriction of virus movement upon inoculation are well documented (Ion-Nagy et al., 2006). This is the reason why, in spite of the large body of literature available, the genetic basis of Sharka resistance is still under debate. From a physiological point of view, different possible candidate genes have been described to be involved in the resistance response to PPV in plants. Among the most interesting ones that can be listed there are the eukaryotic translation initiation factor eIF4E and genes involved in the process of RNA silencing. The eIF4E factor and its isoforms [eIF(iso)4E] have been shown to play a role in the susceptibility or resistance to virus infection in various plant species (Robaglia and Caranta, 2006; Wang et al., 2013). Nevertheless, in most cases, these factors have been shown to account for a recessive inheritance of the resistance trait. In addition, further research has identified a more complex genetic control featuring two or more genes in apricot and related Prunus species (Decroocq et al., 2006; Pilarová et al., 2010; Dondini et al., 2011). A more recent gene expression analysis of Plum pox virus susceptibility/resistance in apricot shows that susceptibility to PPV in apricot is a complex process based on a continuous battle between the virus (PPV) and the plant, both at the pathogen resistance gene level (allene oxide synthase, the S-adenosylmethionine synthetase 2 and the major MLP-like protein 423) and gene silencing level. This was confirmed by transcriptomic differences at the gene expression level. On the other hand, resistance to PPV in apricot is also a complex process that could involve MATH genes (Rubio et al., 2015b). Zuriaga et al. (2013), using a positional cloning approach, also suggested the involvement of MATH genes in the resistance mechanisms, but probably together with other genes. This result was confirmed through a genome-wide association



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study (Mariette et  al., 2016) which speculated on two candidate genes for PPV resistance in apricot: a BTB/POZ-MATH-TRAF-like protein and a MAPK dual-specificity phosphatase. For the first gene, in Arabidopsis, another MATH-TRAF was demonstrated to control long-distance movement of potyviruses, that includes PPV but, in this case, the candidate gene is a coiled-coil MATH-TRAF-like protein (Cosson et al., 2010). Decroocq et  al. (2014) developed a single length polymorphism (SSLP) marker called ZP002, which is based on the 5-bp deletion present in the putative gene ppb0022195 which encodes a MATH-TRAF-like protein, mapping at position 8,157,652 (relative to version 1.0 of the peach genome). Following the identification of such alleged resource of resistance, the investigation on the MATH gene(s) as a promising candidate in apricot has spurred the search of quick and cost effective methods to early genotype accessions for resistance. In order to develop new tools to understand the genetic control of PPV resistance in apricot, Savazzini et al. (2017) constructed a BAC library that covers around 10× the ‘Lito’ cultivar haploid genome. ‘Lito’ is a resistant parent that has been used to address the segregation of the trait in controlled crosses (Dondini et al., 2011) in order to identify candidate genes for Sharka resistance. In this work, new SSRs markers covering the linkage group 1 (LG1) of cultivar ‘Lito’ were used for the construction of a minimum tiling path of BAC clones covering the Sharka resistance region. Recently, a method has been designed that exploits the high-resolution melt (HRM) real-time PCR analysis specifically targeting the 5-bp deletion described in the ppb0022195 gene above mentioned (Passaro et al., 2017). The wide application of such methods can foster marker-assisted selection (MAS) projects to introgress the resistance allele(s) of diverse resistant cultivars into new commercial breeds. For the second gene, members of this family are known from previous studies to play roles in pathogen resistance (Gupta et al., 1998). Castelló et al. (2010) demonstrated the role of a DNA-binding protein phosphatase, DBP1, in Arabidopsis infection by PPV. However, it does not belong to the same class of phosphatases as the one found in apricot. The GWA study might indicate either a new role in viral susceptibility or resistance for a protein phosphatase distinct from DBP1 or that the gene found in apricot is tightly linked to the true (but still unknown) gene controlling resistance to Sharka, but further experiments are needed to test these hypotheses.

10.2.2  Transcriptional studies New approaches, such as next-generation RNA sequencing (RNA-seq), have recently been exploited in order to achieve better understanding of the plant responses to PPV. Rodamilans et al. (2014) compared PPV-infected and non-infected samples by RNA-seq in order to study the hypersensitive response to PPV in the European plum ‘Jojo’ (Prunus domestica L.). This study allowed to identify 3020 unigenes that are differentially expressed during infection, 154 of which are related to genes implicated in defense responses. Another study was performed by Rubio et al. (2015a) to better understand PPV susceptibility in peach (P. persica L. Batsch). RNA-Seq results showed that the nature of the peach-PPV interaction at the transcriptome level is dynamic. Early PPV infection in peach leaves without symptoms is associated with an induction of genes related to pathogen resistance, such as jasmonic acid, resistance proteins, chitinases and Lys-M proteins. On the other hand, when the

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virus is fully installed, the overexpression of Dicer protein 2a genes could be representative of a gene silencing response suppressed by the HC-Pro and P1 PPV proteins. To contribute to knowledge in the field, Rubio et  al. (2015b) analyzed gene expression changes in leaves from two full-sib apricot genotypes, resistant ‘Rojo Pasiòn’ and susceptible ‘Z506–7’ in response to PPV infections. In general, infected apricot leaves have a low representation of genes involved in the “cellular process” and a high representation of genes implicated in “catalytic activities” and the “regulation of metabolic and biological processes”. The study confirmed also that susceptibility to PPV in apricot is a complex process based on a continuous battle between the virus and the plant at the pathogen resistance gene level (allene oxide synthase, the S-adenosylmethionine synthase 2 and the major MLP-like protein 423) and gene silencing level as observed in peach. On the other hand, resistance to PPV is also a complex process in which could involve MATH genes as reported above but also other genes like Pleiotropic drug resistance 9 gene, Cysteine-rich secretory proteins, Antigen 5, Pathogenesis-related 1 protein and Late embryogenesis abundant protein. More recently, Zuriaga et  al. (2018) combined transcriptomic and genomic data to hypothesize that PPV resistance in apricot is associated with down-regulation of two MATHd genes. In particular, the work speculates that one MATH gene called ParPMC2 could be a pseudogene that mediates down-regulation of its functional paralog ParPMC1 by silencing. Further functional analyses are necessary to elucidate the role of these genes and to identify additional host S-genes involved in viral infection.

10.3  Molecular tools to diagnose PPV and test resistance in planta Plants show different symptoms of the viral infection on leaves, stems and fruits (Fig. 10.2, López et al., 2003; Al-Hiary et al., 2011). Early detection of plant pathogens allows to manage infections in greenhouse systems and in the field during different stages of disease development; in addition, it could be possible to minimize the risk of the spread of infections as well as to prevent the introduction of new plant pathogens, especially quarantine pathogens. In fact, the EU quarantine regulation (Directive 2000/29) enforces mandatory controls either for the declared new foci monitoring as well as on the post-entry consignments of Prunus plants for planting. Despite the visual inspection of symptoms represents an useful first measure of diagnosis, it fails to detect the presence of pathogen in the early infection stages, when they are symptomless. Moreover, virus presence in the plant is often uneven and dependent on the developmental stage and climatic conditions. In this perspective, the availability of a quick, sensitive and inexpensive diagnostic tool to screen even dormant propagative material is highly valuable. In addition, rapid and reliable diagnostic methods can serve as a measure to early phenotype plant resistance in a large number of individuals at relatively low cost and labor, thus assisting the creation of new élite cultivars through both conventional and markerassisted breeding (MAB). At the early stages of PPV investigation Kerlan and Dunez (1979) differentiated serologically the D (Dideron) and M (Marcus) strains, the former on apricot in France and the latter originally on peach in Greece. In the later decades, with the extensive setting of molecular biology tools, alternative and more sensitive ways to detect virus in plants have been developed. Nowadays, when dealing with molecular diagnostics in the field, a combination of serological and molecular



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t­echniques is routinely employed (Sochor et al., 2012; García et al., 2014; Clemente-Moreno et al., 2015). Techniques of the first group are based on the use of antibodies specific to the pathogen antigens and include enzyme-linked immunosorbent assay (ELISA), immunoblotting and lateral flow (LF). Immunoassay technology with monoclonal antibodies allows the detection of plant viruses with a high specificity, resulting ideal for high-throughput testing plant samples and for the on-site detection of plant pathogens, as done with tissue print ELISA and related devices. Novel approaches, like those based on immunochromatography (Maejima et al., 2014) or electrochemical impedance spectroscopy (EIS) (Ambrico et al., 2016; Jarocka et al., 2011), have been recently reported. On the other side, nucleic acid-based methods are more accurate and specific enough to detect a single target pathogen within a complex mixture containing more than one analyte and they are highly effective to detect multiple targets. Typically, methods like real time RT-PCR and its various declinations, offer the best performances as of sensitivity and accuracy although they suffer of lack of portability that limits in-field adoption. Adaptation and optimization of the molecular techniques allowed to develop rapid, onestep real-time fluorescent-based detection of PPV (Mavrodieva and Levy, 2004). Nonetheless, various limitations affect the proper assessment of virus load and the progression and state of infection in stone fruit species. In particular, fruit trees have to be grown in pots and with alternate cycles in greenhouses or cold chambers; this means a limitation of the period during the year that is suitable for the artificial transmission of PPV, the low concentration and irregular distribution of PPV throughout the plant, the environmental influence on PPV symptom evolution and at least two vegetative periods needed to score the reaction of plants following infection (Rubio et al., 2009). In addition, when working with transgenic material and a quarantine virus, the evaluation process needs to respect restrictive and expensive control measures (García-Almodóvar et al., 2014). In order to easily identify PPV resistant plant material, in vitro virus inoculation procedures have been devised by grafting the transgenic apexes on PPV-infected ‘GF305’ rootstocks (Lansac et al., 2005; Monticelli et al., 2012). In spite of these advantages, molecular detection methods suffer some limitations when dealing with pathogens at low titres like at early infection stages or in matrices such as seeds and insect vectors. Moreover, false negative results can be produced from cross contamination with PCR components which can completely interfere with amplification of target DNA, while false positive results can be generated by cross-amplification of off-target DNA fragments. Another limitation is related to the inability to apply PCR-based methods for plant pathogen detection in the field. In the last years, technologies have emerged which are based on the rapid isothermal amplification for diagnostic purposes in plant pathology (Kim and Easley, 2011). Zhang et al. (2014) have reported the development of isothermal AmplifyRP® that does not need any sophisticated or costly equipment for rapid detection of PPV in woody crops under both laboratory and field conditions. Unlike the human health field, where it is a well established practice, the concept of ‘point of care’ (POC) applied to virological diagnostics in plants is only recently emerging. The early and cost-effective detection of pathogens in cultivated plants can reduce dramatically the time and cost associated to PPV determination in the field and control the pathogen spread in the orchards. An example of application with potyviruses in Prunus species is provided by the recent work of Silva et al. (2018).

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Cutting-edge technological improvement and the concurrent reduction in the costs of mixed organic- and electronic-based methods are foreseen to revolutionize the way diagnostics will be done in the next years. Among these, electrochemical DNA biosensors for plant pathogen determination are based on label-based and label-free voltammetric detection of DNA hybridization. In addition, among the newest approaches, those based on DNA translocation through nanopores, show a great potential in this field. Label-free DNA hybridization has been exploited using amperometric, impedimetric and voltammetric detection including square-wave voltammetry (SWV), cyclic voltammetry (CV) and differential pulse voltammetry (DPV). For an extensive coverage of these methods see the excellent review of Khater et al. (2017). An interesting example of voltammetric approach has been recently reported for the label-free detection of picomolar concentrations of nucleic acid from Plum pox virus (PPV) using glassy carbon electrodes. They exploited the Osteryoung square wave voltammetry (OSWV) technology to detect the pathogen-related DNA and the complementary target immobilized on the electrode (Malecka et al., 2014). Organic electronic based devices are emerging in the field of biosensing and they appear to be promising candidates to establish quick, inexpensive and sensitive diagnostic tool to screen even dormant propagative material. Technically, they are based on both the Electrolyte Gated Organic Field Effect Transistor (EGOFET) and the Organic Electrochemical Transistor (OECT) principles (Wang et al., 2015; Rivnay et al., 2018). They offer a unique combination of advantages like the use of organic electronic materials which warrant low production costs in environmentally friendly conditions and the adoption of flexible substrates. In addition, the amplification property inherent to the Field Effect Transistor (FET) principle, allows to attain extremely low limits of detection (Simon et al., 2016; Wang et al., 2016). An interesting application of this technology has been demonstrated for the detection of PPV in plant extracts with a sub ng/ml detection limit. The proof-of-principle sensing unit of the biosensor is based on anti-PPV antibodies coupled to the electronic amplification system which can easily be reconfigured into a handy tool for greenhouse and field operation (Berto et al., 2019).

10.4  Strategies to restrict virus spread 10.4.1  Transgenic protection Various transgenic strategies have been adopted to produce stone fruits species resistant to PPV. They span from the traditional coat protein (CP)-based approach to the use of alternative target genes. It is not the aim of this chapter to discuss them as excellent reviews already exist (Ilardi and Tavazza, 2015; Limera et al., 2017). For sake of convenience a list of genes already tested or candidates to be exploited to confer resistance or tolerance, based on the most recent literature, is made available in Table 10.1. In particular, we need to draw the reader's attention to those that are emerging from novel trends of investigation like the control of aphid vectors or the modulation of plant physiological response following PPV infection. What should be remarked here is the scarcity of transformation and/or regeneration methods available for most stone fruit species which still strongly impairs the possibility to harness the potential of various (candidate) genes to both study their function and behaviour in these species and



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TABLE 10.1  List of genes and strategies used to control PPV virulence and spread. Gene

Plant species tested

Mechanism of action

Literature source

Coat protein (CP)

Plum

RNA silencing

Scorza et al. (1994, 2001, 2013); Sidorova et al. (2018)

NBS-LRR genes

Apricot

Plant-pathogen recognition

Soriano et al. (2005)

Argonaut AGO1 protein

Arabidopsis

RNA silencing

Sicard et al. (2008)

RNA helicase-like protein, AtRH8

Arabidopsis

Virus diffusion in planta

Huang et al. (2010)

Potyvirus nuclear inclusion proteins (NIa and NIb)

N. benthamiana

RNA silencing

Guo et al. (1998); Gil et al. (2011)

Eukaryotic translation initiation factor 4E isoform (eIF(iso)4E)

Peach

Host translation machinery

Cui and Wang (2017)

RTM/MATH genes

Arabidopsis, Apricot

Virus diffusion in planta

Decroocq et al. (2006); Cosson et al. (2010); Zuriaga et al. (2013)

P1

Plum

Suppression of RNA silencing

Di Nicola-Negri et al. (2005); García-Almodóvar et al. (2014)

HC-Pro

Tomato, Peach, Plum

Suppression of RNA silencing

Di Nicola-Negri et al. (2005); García-Almodóvar et al. (2014); Gogoi et al. (2017)

Toll-like receptor 7 (TLR7)

Tobacco, Bemisia tabaci

Control of aphids

Chen et al. (2015)

MpC002, Rack-1

N. benthamiana, Arabidopsis

Control of aphids

Pitino et al. (2011)

Salicylic acid (SA)

Peach

Phytohormone signaling

Dehkordi et al. (2018)

to exploit them for agronomic purposes. In this regard, only few cases were reported with limitation to few genotypes (Petri and Burgos, 2005; Petri et al., 2008).

10.4.2  RNA silencing Various molecular types fall under the class of small RNAs (sRNAs) including microRNAs (miRNAs) and small interfering RNAs (siRNAs) that target complementary mRNAs or DNA modulating post-transcriptional silencing (PTS) or transcriptional silencing of the corresponding target genes (Khalid et  al., 2017; Zhao and He, 2018). RNAi plays a crucial role in defense against viruses in plant and animal kingdom, triggering the RNA degradation sharing sequence similarity to a specific dsRNA. The pathway leading to this degradation has emerged as a powerful strategy to engineer transgenic disease resistance against pests and pathogens in plants (Gordon and Waterhouse, 2007; Lilley et al., 2012; Koch et al., 2013; Duan et al., 2012).

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Plants use RNA silencing as a natural immunity mechanism as one of their defense strategies to restrict pathogen invasion (Ding, 2010), and many viruses have evolved to express VSR proteins to counter host antiviral RNA silencing (Burgyán and Havelda, 2011; Duan et al., 2012). Ahead of the description of the sequence-dependent RNA silencing mechanism in 1998 (Voinnet et al., 1998), virologists had observed that the expression of viral coat protein (CP) in transgenic plants made them resistant against infection by the homologous virus. This kind of pathogen-derived resistance (PDR), that was also named protein-mediated resistance, has been described in various virus groups including the potyvirus one (Abel et al., 1986; Beachy et al., 1990; Kawchuk et al., 1990; Lindbo and Dougherty, 1992a,b; Jan et al., 1999). Since then, virus resistance has been attempted using other viral proteins including movement protein (Sijen et  al., 1996), replication-associated protein (Canto and Palukaitis, 1998; Chellappan et  al., 2004), the potyvirus nuclear inclusion proteins (NIa and NIb) (Guo et  al., 1998), viral suppressor of RNA silencing (Pruss et al., 2004; Fagoaga et al., 2006; Germundsson and Valkonen, 2006), plus some other viral proteins (Germundsson and Valkonen, 2006; Bucher et al., 2006). The plum pox virus proteins HC-Pro and P1 act by suppressing RNA silencing (Pruss et al., 1997; Kasschau and Carrington, 1998); they are the first two open reading frames products of the PPV genome and act by processing themselves at their respective C-termini (Lopez-Moya et al., 2000; Di Nicola-Negri et al., 2005). Cui and Wang (2017) first have demonstrated the development of a viral vector derived from Prunus necrotic ringspot virus (PNRSV), a widespread fruit tree virus endemic in all Prunus fruit production countries and the use of such vector to silence endogenous genes in peach. In particular, they used it to silence the eukaryotic translation initiation factor 4E isoform (eIF(iso)4E), a factor that many potyviruses, including Plum pox virus (PPV), deploy to tackle the host translation machinery. Interestingly, the eIF(iso)4E-knocked down peach plants were resistant to PPV. This approach proves a potential new route to control virus diseases in perennial trees via viral vector-mediated silencing of host factors. In addition, the PNRSV vector might serve as a powerful molecular tool for functional genomic studies of Prunus fruit trees. A complex and coordinated network of functions is necessary for the movement of RNA silencing signal molecules within a plant. Following the synthesis, sRNAs require loading into the vascular system, translocation and unloading into the target tissues (Melnyk et al., 2011; Voinnet et al., 1998). Depending on the source and sink tissue variable times, from hours to days, can be necessary to accomplish such a long distance transport of signal molecules (Voinnet et al., 1998; Konakalla et al., 2016). At the intersection between intra- and inter-cellular signaling and networking a major role in eukaryotes is played by the conventional secretory pathway which secretes proteins containing signal peptides and other contents via fusion of secretory vesicles with the plasma membrane (PM). Extracellular vesicles (EVs) are emerging more and more as a new way of cross-kingdom trafficking that plays an important role in plant–pathogen interactions (Rutter and Innes, 2018; Cai et al., 2018), in particular between plants and pathogenic microbes (Gogoi et al., 2017). A recent report has demonstrated that extracellular vesicles act as carriers of sRNA entities for plant disease resistance (Cai et al., 2018). Although the first reports of plant EVs date back to the late 1960s, their function and composition remain poorly understood (Rutter and Innes, 2018). In the first study to isolate and



10.4  Strategies to restrict virus spread

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purify plant EVs from leaves of the model species Arabidopsis it has been revealed that plant EV production is enhanced in response to biotic stress and that they are enriched for defense/stress-related proteins (Rutter and Innes, 2016). In this study the EV proteome also was enriched for various membrane-trafficking proteins. Interestingly, one of these proteins, SYP71 is localized mainly to the endoplasmic reticulum and it is necessary for the infection of Arabidopsis by the potyvirus-type Turnip mosaic virus, where it plays a role in the fusion of virus-induced vesicles with chloroplasts (Wei et al., 2013). The involvement of EV in plantvirus interaction is a promising territory of investigation that deserves attention and whose results will advance both the knowledge of the mechanisms involved and provide means to control viral infections. An alternative way to interact with the plant defense machinery, of special interest for tree species, is represented by the mechanic delivery of interfering RNA molecules through holes drilled in the trunk or by petiole absorption as it was demonstrated for two agronomically important fruit species, apple and grapevine (Dalakouras et al., 2018). Although a proof of principle, this approach paves the way to extensive experimentation with tree species which are typically more difficult to transform.

10.4.3  Spray-induced gene silencing (SIGS) A very attractive alternative to virus resistant transgenic crops or to the use of pesticides against virus aphid vectors is represented by the exogenous application of double-stranded RNA (dsRNA) (Tenllado and Díaz-Ruíz, 2001; Tenllado et al., 2003; Gan et al., 2010; Lau et al., 2014; Wang and Jin, 2017). This approach is even more attractive on species for which consistent protocols for transformation and regenerations are not available, like most stone fruit trees (Burand and Hunter, 2013; Revers and García, 2015; Robinson et al., 2014). Nevertheless, spraying dsRNA onto plants proved challenging as protection against homologous viruses is provided for a limited time due to the instability of the naked dsRNA (Mitter et al., 2017a). A typical virus protection window of 5–7 days post spray has been observed (Tenllado et al., 2003; Gan et al., 2010). The mechanism of entry, translocation and processing of exogenously applied dsRNA in plants is still poorly understood. Anyway, the persistence of dsRNA artificially supplied to plant tissues can last enough to be transmitted to predators. For instance, it has been shown that plant insects and mites can uptake double-stranded RNA upon its exogenous application on tomato leaves using the Zucchini yellow mosaic virus (ZYMV) model. In this study the viral HC-Pro region was targeted using tomato plants and the corresponding dsRNAs were detected in aphids (Myzus persicae), whiteflies (Trialeurodes vaporariorum) and mites (Tetranychus urticae) that were fed on treated as well as systemic tomato leaves (Gogoi et al., 2017). As an example, transient and stable expression of dsRNAs in transgenic model species (N. benthamiana and Arabidopsis, respectively), can reduce progeny population by silencing aphid genes (Pitino et al., 2011). Also, expression of dsRNA corresponding to Toll-like receptor 7 (TLR7) gene of whitefly (Bemisia tabaci) in recombinant entomopathogenic fungi (Isaria fumosorosea) successfully downregulated the target gene expression, resulting in enhanced whitefly mortality (Chen et al., 2015). Notably, being Myzus persicae also a major vector of sharka disease in Prunus species, this evidence strongly supports the possible role of exogenous or endogenous dsRNA species to both control plant pests and restrict virus propagation in the orchard.

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Artificial dsRNA delivery, apart from its obvious agricultural use, could also prove a useful mean for the functional characterization of PPV insect vector's genes. Cutting edge approaches, such as the use of nanoparticles as carriers of dsRNA to enhance stability and sustained release, are emerging as innovative technologies. Among the products tested there are reports on the BioDirect technology from Monsanto as an RNAi spray application, yet the details of this technology have not been disclosed in detail. A number of questions remain regarding how to deliver large dsRNA fragments into plant cells, the mechanism of dsRNA uptake into plant cells, and the stability of the topically applied dsRNA to withstand environmental conditions and provide long-term protection against the targeted virus. The practical adoption of this technology will depend also an the sustainability of costs associated to the synthesis of dsRNA molecules for application at a greenhouse or field scale. Up to now, the main approach for producing dsRNAs has been physical annealing of two enzymatically synthesized ssRNA strands with annealing performed either in vitro or in vivo. A recent alternative has been suggested that exploits the biosynthetic ability of a bacteriophage-derived machinery to efficiently synthesize dsRNA and its possible application to transform plants providing an inborn ability to produce the triggering molecules to elicit resistance (Niehl et al., 2018). In addition, the combination of novel methods to convey dsRNA molecules has been demonstrated to extend to at least 20 days the persistence of the triggering stimulus on the leaf surface (Mitter et al., 2017b). The adoption of proper regulations will also establish the way such a technology could be applied.

10.5 Conclusions Since the first successful application of PDR in creating virus-resistant plants, a number of strategies have been developed based on the mechanism. A better understanding of RNA silencing pathways has also contributed to the development of this technique. The RNA silencing-mediated approach is now a powerful tool in antiviral research, backed by the promising results obtained in the HIGS-mediated fight against bacterial and fungal pathogens and insect pests. Although RNA silencing has been successful, there are still many limitations in utilizing this strategy. RNA silencing-mediated resistance and the silencing efficacy are the results of interaction between many factors, including sequence similarity, target selection, pathogen titer, and environmental temperature (Szittya et  al., 2003). Thus, it is difficult to accurately predict the resistance efficacy. Moreover, most of the successful examples were obtained in greenhouses. Considering that mixed infections are common in nature, it is still a challenge to obtain resistant plants. Therefore, further scientific research is required to uncover the factors affecting RNA silencing-mediated resistance in specific cases and to test the resistance efficacy in the field. The investigation on the mechanisms of PPV resistance is still hindered by the various factors controlling the virus cycle. Nevertheless, the efforts to comprehend its biology and overcome its diffusion are continuous and the contribution of the modern non-transformative technologies could represent a major tool in the toolbox to control and, possibly, eradicate sharka in the future.

References 217

10.6  Data repositories and information mining tools Several online resources are available that collect and distribute information concerning Plum pox virus and Sharka to those interested to further the knowledge about this disease. A list is provided below (all sites are updated at December 2018). • The Centre for Agriculture and Bioscience International (CABI) web page on PPV: https://www.cabi.org/isc/datasheet/42203 • The American Phytopathological Society (APS) page on PPV: http://www.apsnet.org/ publications/apsnetfeatures/Pages/PlumPoxPotyvirus.aspx • The Plum Pox Virus database: http://w3.pierroton.inra.fr:8060/users/login • The official page of the UE-funded “Sharka Containment (SharCo)” project: http://www. sharco.eu/ • The PPV diagnostic protocol adopted by the Seventh Session of the Commission on Phytosanitary Measures: https://www.sharco.eu/content/download/3350/35921/ version/1/file/ISPM+27+Diagnosttic+protocols+DP2+PPV.pdf • The site of the Genome Database of Rosaceae (GDR) with various resources on the Prunus species genomes, molecular markers and maps available: http://www.rosaceae.org • The European Plant Protection Organization (EPPO) Global Database (GD) entry on PPV: https://gd.eppo.int/taxon/PPV000

Acknowledgments Authors wish to thank Prof. Raffaele Testolin, University of Udine, for the critical suggestions on the manuscript.

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Zhang S, Ravelonandro M, Russell P, McOwen N, Briard P, Bohannon S, Vrient A. Rapid diagnostic detection of plum pox virus in Prunus plants by isothermal AmplifyRP(®) using reverse transcription-recombinase polymerase amplification. J Virol Methods 2014;207:114–20. Zhao QY, He XJ. Exploring potential roles for the interaction of MOM1 with SUMO and the SUMO E3 ligase-like protein PIAL2 in transcriptional silencing. PLoS One 2018;13:e0202137. Zuriaga E, Soriano JM, Zhebentyayeva TN, Romero C, Dardick C, Cañizares J, et al. Genomic analysis reveals MATH gene(s) as candidate for Plum pox virus (PPV) resistance in apricot (Prunus armeniaca L.). Mol. Plant Pathol 2013;13:663–77. Zuriaga E, Romero C, Blanca JM, Badenes ML. Resistance to Plum Pox Virus (PPV) in apricot (Prunus armeniaca L.) is associated with down-regulation of two MATHd genes. BMC Plant Biol 2018;18:25.

Further reading Abbott A, King GJ, Iezzoni AF, Arùs P. A set of simple-sequence repeat (SSR) markers covering the Prunus genome. Theor Appl Genet 2003;106:819–25. Borges F, Martienssen RA. The expanding world of small RNAs in plants. Nat Rev Mol Cell Biol 2015;16:727–41. Dondini L, Lain O, Geuna F, Banfi R, Gaiotti F, Tartarini S, Bassi D, Testolin R. Development of a new SSR based linkage map in apricot and analysis of synteny with existing Prunus maps. Tree Genet Genomes 2007;3:239–49. Hadidi A, Flores R, Candresse T, Barba M. Next-generation sequencing and genome editing in plant virology. Front Microbiol 2016;7:1325. Karayiannis I, Thomidis T, Tsaftaris A. Inheritance of resistance to Plum pox virus in apricot (Prunus armeniaca L.). Tree Genet Genomes 2008;4:143–8. Katiyar-Agarwal S, Jin H. Role of small RNAs in host-microbe interactions. Annu Rev Phytopathol 2010;48:225–46. Wang M, Thomas N, Jin H. Cross-kingdom RNA trafficking and environmental RNAi for powerful innovative preand post-harvest plant protection. Curr Opin Plant Biol 2017;38:133–41.