Introduction
Sharka is the most dangerous viral disease in trees of
the Prunus genus, causing substantial economic losses (Cambraet al. , 2006). The etiological agent is the Potyvirus Plum
pox virus (PPV) which is naturally transmitted by aphids in a
non-persistent manner and by grafting PPV-infected material.
PPV has great genetic diversity. PPV isolates can be subdivided into ten
strains based on phylogenetic analyses (Hajizadeh et al. , 2019;
Rodamilans et al. , 2020). Among these strains, the two most
widespread and economically significant are Dideron, PPV-D and Marcus,
PPV-M, (García et al. , 2014). In particular, PPV-D is endemic in
Spain. Symptoms induced by PPV on apricot include chlorotic bands and
rings on leaves and stones, fruit deformation, and early fruit drops
devaluating fruits (Agustí, 2010). The virus does not kill the trees but
may largely reduce production (García and Cambra, 2007).
PPV resistance presents one of the most discussed topics in European
apricot (Prunus armeniaca L.) breeding programs (Rubio et
al. , 2008; Krška, 2018). All apricot cultivars of European origin are
susceptible to PPV (Krška, 2018). One major limitation of introducing
PPV resistance in European apricots is that most of the genetic sources
are North American cultivars (Martínez-Gómez et al. , 2000),
characterized by high dormancy requirements and poorly adapted to the
Mediterranean climatic conditions. In this context, exploring
alternative strategies to confer PPV resistance is essential.
Among the transgenic strategies used to induce PPV resistance,
significant results have been obtained through the biotechnological
exploitation of RNA silencing (reviewed in Ilardi & Tavazza, 2015). RNA
silencing is a sequence-specific gene-regulation mechanism widely
conserved among eukaryotes. In plants, among other functions, RNA
silencing exerts a pivotal defense role against viruses and viroids.
Double-stranded RNA (dsRNA) is the crucial trigger for RNA silencing.
Dicer, a ribonuclease (RNase) III family, is involved in the cleavage of
the dsRNA into 21–24 nucleotide (nt) duplexes, referred to as short
interfering RNAs (siRNAs). In the case of Post-Transcriptional Gene
Silencing (PTGS), siRNAs loaded into Argonaute proteins guide them to
slice and/or translational repress complementary RNA sequences (reviewed
in Zhao and Guo, 2022). Therefore, transgenic expression of a
viral-derived dsRNA has been proven to be a robust strategy to confer
virus resistance in crops (Smith et al. , 2000). RNA silencing can
spread to neighboring cells through plasmodesmata or systemically
through the vascular system (Mlotshwa et al. , 2002; Palauquiet al. , 1997). Thus, RNA silencing induced in a restricted tissue
of the plant can potentially spread to other tissues. The nature of the
mobile RNA silencing signals remained debated until two complementary
works shed light on it (Dunoyer et al. , 2010; Molnar et
al. , 2010). Using different approaches, they showed that siRNAs are
indeed the mobile signal. The evidence that RNA silencing can move over
long distances through the vasculature opened the question of whether
virus resistance of a transgenic rootstock could be transmitted to a
non-transgenic scion through grafting, thus overcoming the concern about
the spreading of transgenes in the environment and eating transgenic
products (Arpaia et al. , 2020).
Grafting has been traditionally used to join scions and rootstocks of
fruit trees with different genomes. In apricot, as in most fruit trees,
it is a common practice for vegetative propagation of commercial
cultivars. Additionally, it is commonly employed for horticultural crops
such as tomatoes or cucurbits to improve productivity (Melnyk and
Meyerowitz, 2015).
The early evidence on the applicability of transgenic rootstock:wt scion
(TR:WS) grafting to confer viroid resistance came from the work of Kasaiet al. , (2013). They showed that genetically modified tobacco
rootstocks expressing Potato spindle tuber viroid (PSTVd) siRNAs
could attenuate PSTVd accumulation in a non-genetically modified tobacco
scion grafted on the stock. In subsequent work, wild-type tomatoes
partially resistant to Cucumber mosaic virus (CMV) were obtained
after grafting them onto transgenic tomatoes transformed with plant
vectors expressing intron-spliced hairpin RNA (ihpRNA) designed to
silence different CMV genes (Bai et al. , 2016). Similarly, Zhao
and Song, (2014) showed that Prunus necrotic ring spot virus(PNRSV) hpRNA-derived siRNAs from the transgenic cherry rootstocks could
confer a certain degree of resistance to the non-transgenic sweet cherry
scions. However, despite this encouraging evidence,
contradictory results were also
obtained in herbaceous and woody plants (Leibman et al. , 2015;
Sidorova et al. , 2021). In particular, Sidorova et al. ,
(2021) showed that although the transgenic rootstocks of the
interspecific Elita cv. [(Prunus pumila L. × P. salicinaLindl.) × (P. cerasifera Ehrh.)] accumulate a high level of PPV
coat protein (CP) siRNAs, the trans-grafting was not successful in
promoting PPV resistance in non-transgenic scions.
However, besides transgenic constructs using PPV CP sequences,
significant results were obtained with the h-UTR/P1 construct, which
encodes an ihpRNA encompassing the first 733 nt of the PPV-M ISPaVe44
genome (Di Nicola-Negri et al. , 2005). In model plants, h-UTR/P1
induced long-lasting PPV resistance not only to the homologous ISPaVe44
isolate but also to isolates belonging to D, M, Rec, and the distantly
related EA and C PPV strains (Di Nicola-Negri et al. , 2005; Di
Nicola-Negri et al. , 2010). In addition, authors showed that PPV
resistance was maintained in transgenic Nicotiana
benthamiana plants under biotic and abiotic stresses (Di Nicolaet al. , 2014). Notably, when the h-UTR/P1 construct was
introduced in the plum, 70% of clones were resistant to PPV in in-vitro
and greenhouse (García-Almodovar et al. , 2015). These encouraging
data prompt us to determine if
the resistance to sharka could be transferred from the transgenic plum
rootstocks to wild-type apricot scions grafted onto them. To this end,
we conducted grafting experiments of wild- type apricots onto the
transgenic plum rootstocks and studied if the resistance was transmitted
from the rootstock to the scion.