Discussion
The success of transgene RNA silencing transmission through grafting
would be of practical importance in horticulture. Grafting wild-type
scions onto transgenic silenced rootstocks could improve individual
traits of well-established non-transgenic tree cultivars, particularly,
for those recalcitrant to regeneration or transformation. It would be
instrumental in the case of Prunus species that are difficult or
still impossible to transform, such as apricot (Petri et al. ,
2015) or peach (Ricci et al. , 2020). Notably, it is expected that
public concerns about using transgenic plants should be mitigated by the
lack of transgenes spreading by outcrossing coupled with the consumption
of non-transgenic edible parts of the plant.
Although encouraging, our first data (Table 1) studying the transmission
of PPV resistance from transgenic plum rootstocks to wt apricot scions
were limited in broadness due to the reduced sprouting of the wt apricot
PPV-infected buds, possibly due to a high inoculum pressure. A similar
technical limitation was also reported by Zhao and Song, (2014), which
showed that grafting PNRSV infected buds onto sweet cherries results in
the death of the buds.
To overcome the above limitation, we adopted the chip budding technique.
This inoculum procedure permitted us to study the resistance behavior of
one hundred and ten grafted wt apricots challenged with a PPV-D isolate.
The data clearly shows an increase with time in the number of PPV-free
plants as evaluated by the RT-PCR analysis (Figure 1). In addition, the
low percentage of PPV-infected apricot plants grafted onto resistant
rootstocks accumulated significantly fewer amounts of the virus when
compared to the susceptible rootstock (Figure 2). Notably, when
non-transgenic scions that recovered from viral infection were
re-challenged with PPV, the RNA silencing effectively eliminated the
virus within the same growth cycle in about 50% of scions (Table 2).
The ability to recover from virus infection is a peculiar characteristic
of the RNA silencing-mediated resistance occurring both during natural
viral infections (Covey et al. , 1997; Ratcliff et al. ,
1997) and in herbaceous and woody transgenic plants (Dougherty et
al. , 1994; García-Almodovar et al. , 2015; Ravelonandro et
al. , 1993; Lindbo and Dougherty, 2005).
To the best of our knowledge, there are only three previous papers
intending induced silencing from a rootstock to a scion in woody plants,
with contradictory results. In apples, transgenic rootstock-mediated
silencing in the scions was shown to occur for a gusA transgene
but not for an endogenous anthocyanidin synthase gene. Additionally, nor
the transgene nor the endogen was silenced when the grafting experiment
was conducted in the greenhouse (Flachowsky et al. , 2012).
Authors hypothesized that lignification might influence cell-to-cell
transport of siRNAs in living cells, thus explaining the lack of
silencing effect.
In a recent paper (Sidorova et al. , 2021), two PPV-resistant
transgenic plum cultivars transformed with a hairpin to silence the
virus capsid gene were evaluated for their capacity to transfer the PPV
resistance character to the wild-type grafts. They found that transgenic
rootstocks remained virus free but could not protect the scion due to
the lack of an efficient transfer of transgene-derived siRNAs from the
rootstocks to the scions. However, scions accumulated specific endogen
sRNAs characteristic of the rootstocks (Sidorova et al. , 2021).
Conversely, Zhao and Song, (2014) showed that PNRSV-hpRNA-derived siRNAs
were transmitted up to 1.2 m from the transgenic sweet cherry rootstocks
to the non-transgenic scions conferring enhanced PNRSV resistance.
Variable results were also obtained in works dealing with
grafting-mediated virus resistance in horticultural species. Baiet al. , (2016) showed that 66.7% to 83.3% of non-transgenic
tomatoes were highly resistant to CMV. In tobacco, detached leaves from
scions grafted on transgenic tobacco silenced for the
endogenous NtTOM1 and NtTOM3 genes were shown to
accumulate fewer tobamoviruses than the control plants (Ali et
al. , 2013). Similarly, Nicotiana benthamiana transgenic plants
expressing a hairpin designed to silence PSTVd produced only attenuation
of viral infection (Kasai et al. , 2013). The contradictory data
can be attributed to differences between plant species, the transgenic
construct used and/or the targeted sequence (exogenous infecting virus
or endogenous gene transcripts).
Our data do not support the hypothesis of lignification as the primary
cause of the lack of RNA silencing spreading from rootstocks to scions
(Flachowsky et al. , 2012; Sidorova et al. , 2021). Apricot
scions and plum rootstocks were well-lignified during the four years
that the experiment lasted (from rooting and acclimatizing the
rootstocks, grafting apricots, and infecting them by chip-budding to the
final evaluation).
The siRNAs analyses identified the accumulation of the transgene-derived
PPV UTR/P1 24 nt siRNAs in apricot scions grafted on the PPV resistant
but not onto the susceptible rootstock (Figure 3). In contrast, faintly
amount of 22 nt siRNAs were detected in the apricot scions on resistant
rootstocks but more clearly seen on St5’-7 scions (Figure 3), suggesting
that the 24 nt siRNAs can be: a) preferably transported over a long
distance (Hamilton et al. , 2002; Molnar et al. , 2010); b)
less prone to degradation or; c) less consumed by AGO in the traversed
and recipient cells (Voinnet, 2022). When studying the molecular
mechanisms associated with the resistance to sharka of C5 plum
(’Honeysweet’), 24 nt siRNAs was related to systemic silencing (Kunduet al. , 2008). In particular, they were present only in resistant
C5 plants but not in susceptible ones nor in C5 plants showing middle
sharka symptoms. The evidence that the tolerant and resistant plum
rootstocks could protect the apricot scions, and that 24 nt siRNAs were
only found in these scions but never in those grafted onto susceptible
St5’-7 line, agrees with results found in ‘Honeysweet’ plum.
Different works suggest that all siRNA classes (21, 22, and 24 nt long
siRNAs) are mobile (Devers et al. , 2020), with the 22 nt siRNAs
having a pivotal role in the siRNAs signal amplification and
translational repression (Chen et al. , 2010; Cuperus et
al. , 2010; Wu et al. , 2020). Trans-grafting movement of siRNAs
is not a simple concentration dependent diffusion process, but probably
requires a selective sRNA sorting mechanism and recent studies suggest
that it might be dictated by sRNA biosynthetic pathways, sRNA sizes,
sequence features such as 5’ nucleotide, or selective RNA-binding
protein partners (Kong et al. , 2022). It will be interesting to
evaluate the amounts and nature/diversity of 5′-nucleotide
identities/sizes of siRNAs accumulating in the grafted apricot scions
and transgenic rootstocks using a more sensible and specific technique.
Northern blot analysis identified, in addition to the siRNA in
PPV-resistant plum, transgene-derived UTR/P1 siRNAs in all transgenic
rootstocks independently on the level of PPV resistance, indicating that
their accumulation is necessary, but not sufficient, to assure efficient
PPV interference. These data agree with those obtained by López et
al. , (2010) in Mexican lime transformed with sense, antisense, and
intron-hairpin cDNAs from viral sequences and with data from tobacco
(Alburquerque et al. , 2012) or plum (Alburquerque et al. ,
2017) transformed with a chimerical transgene designed to silenceAgrobacterium oncogenes iaaM and ipt . In those
works, all resistant lines accumulated transgene-derived siRNAs, but
this was not necessarily associated with resistance to citrus tristeza
virus (López et al. , 2010) or crown gall (Alburquerque et
al. , 2012; Alburquerque et al. , 2017). Therefore, a lower amount
of hpRNA seems to be better correlated with resistance. Although this
could be due to lower expression or higher degradation, it seems logical
to think that resistance is related to a more efficient degradation of
the hpRNA.
Previous studies showed that transgenic C5 plants were resistant to PPV
when exposed to natural viruliferous aphids while accumulating low-level
PPV near the graft junction if graft-inoculated (Malinowski et
al. , 2006). Based on the C5 plants data, we expected that the apricots
grafted onto the PPV-resistant plum lines should also be resistant to
PPV infection under natural field conditions. Importantly, since the
PPV-derived h-UTR/P1 construct present in transgenic plum rootstocks was
derived from a PPV isolate belonging to the M strain while the plants
were challenged with a PPV-D isolate, it suggests that the resistance
observed should be extended to, at the very least, the viral isolates of
the two most important and widespread PPV strains.
As conclusion, the results demonstrate for the first time that
PPV-resistant transgenic plums can effectively confer sharka resistance
in grafted non-transgenic apricots scions. It is expected that
using transgenic rootstocks can
mitigate public concerns about transgene dispersions and eating
transgenic food.
Additional studies on the long-distance movement of the RNA silencing
signal are required to understand how broadly applicable this technique
is to modulate the phenotype of wild-type grafted scions in woody
plants. Uncovering the mechanism of sRNA selection for trans-grafting
transport will potentially enhance success in designing artificial sRNAs
to control plant disease.