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Journal of Experimental Botany Advance Access published October 21, 2014
Review Paper
Key words: Antioxidant machinery, biochemical responses, oxidative stress, physiological responses, Plum pox virus, proteomic
analysis, Prunus, sharka.
Introduction
Sharka disease is considered one of the most detrimental dis- the European continent towards the most important Prunus-
eases affecting many stone fruits and is also among the most growing areas around the world, with the exception of
studied viral diseases in the world (Scholthof et al., 2011). Australia, New Zealand, South Africa, and California (USA)
The causal agent of sharka is Plum pox virus (PPV), belong- (Garcia and Cambra, 2007). PPV has spread over long dis-
ing to the Potyvirus genus within the family Potyviridae. PPV tances through the introduction of infected propagative plant
isolates can be subdivided into at least eight strains based on material followed by local dispersion by aphids in a non-
phylogenetic analyses, although new PPV isolates are contin- persistent manner. Strategies used to control the dispersion
uously being collected worldwide (James etal., 2013; Garca of the disease include the use of certificated PPV-free plant
etal., 2014; lba etal., 2014). Among these strains, PPV D material, periodic surveys of orchards, and the eradication of
and M are the most economically important and widespread diseased trees. The reduction of the aphid vector in orchards
(James etal., 2013; Garca etal., 2014). by insecticide treatment is not effective against non-persistent
During the last several decades, sharka disease has had a viruses such as PPV. Disease control is very difficult, how-
significant agronomic impact and has resulted in major eco- ever, because sharka symptoms are highly dependent on both
nomic losses, affecting mostly the Prunus genus (Cambra environmental conditions (temperature, age of the trees,
et al., 2006). Indeed, since its first description in Bulgaria, etc.) and the sensitivity of the host plant. Typical sharka
in 1917 (Atanassoff, 1932), sharka disease has spread from symptoms include chlorotic spots or rings, vein clearing and
The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
For permissions, please email: journals.permissions@oup.com2014
Page 2 of 11|Clemente-Moreno etal.
distortion on leaves, necrotic areas under shallow pale rings, due to the irregular distribution of PPV in plant tissues.
and deformation on fruits (Fig. 1) (Sochor et al., 2012). In For more information about the diagnosis and detection of
addition, fruits may drop prematurely, reducing both fruit PPV, recent reviews such as those by Sochor etal. (2012) and
quality and yield. In sharka-affected countries, the yield Garca etal. (2014) are suggested.
reduction of infected trees as well as the disease symptoms To date, very few sources of PPV resistance have been
on fruits that make them unmarketable must be added to the identified in stone fruit species. Nevertheless, breeding pro-
costs of control, surveillance, and diagnostic and eradication grammes developed since the 1980s have characterized several
programmes. In 2006, it was estimated that sharka had cost PPV-resistant cultivars from different Prunus species, with
a total of ~10 000 million Euros over the previous 30years most occurring in Prunus armeniaca (apricot) originating
worldwide (Cambra etal., 2006; Barba etal., 2011). from North America (Martinez-Gomez and Dicenta, 2000),
Most of the works on PPV have focused on its biological but also in cherry, almond (Rubio et al., 2005), and more
features, genome organization, vectors, host plants, distribu- recently in plum (Bozhkova and Milusheva, 2013). In peach,
tion, and serological and molecular variability (Sochor etal., however, the most economically important Prunus species, no
2012; James et al., 2013; Garcia et al., 2014). The existing source of resistance has been found in spite of the consider-
research concerning sharka disease includes the following able effort carried out by breeding programmes (Rubio etal.,
three main topics: rapid and accurate diagnosis; the identi- 2012). In the absence of resistant cultivars, tolerant cultivars,
fication of sources of resistance; and the obtention of new which display an active defence response resulting in local-
PPV-resistant varieties through classical or biotechnological ized cell death and symptomless fruit, have been used in some
breeding techniques. The diagnostic methods have included breeding programmes (Hartmann, 1998). The incorporation
graft inoculation in GF305 peach (Martinez-Gmez and of resistance genes from other related wild species has also
Fig.1. Sharka symptoms in the leaves of PPV-infected (A) peach, (B) apricot, (C) plum, and (D) pea. (This figure is available in colour at JXB online.)
Plant responses to sharka | Page 3 of 11
useful for breeding programmes. New molecular mark- and DHAR activities was recorded in comparison with
ers would help to select PPV-resistant sources. Rubio etal. non-inoculated (control) plants. Based on the different
(2014) described simple sequence repeat (SSR) resistance behaviour of SOD (H2O2-generating enzyme) and APX
markers linked to PPV resistance. These authors suggest that (H2O2-scavenging enzyme) in both cultivars, the authors
the use of homozygous resistant parents for SSR alleles with suggested a role for H2O2 in the response to PPV in the
good agronomic characteristics would improve the efficiency resistant cultivar (Hernndez etal., 2001). In fact, a dual
of breeding programmes. However, other authors suggest role for H2O2 has been widely described, as it is toxic in
that marker-assisted breeding results could not be sufficient high concentrations, whereas in low concentrations it acts
to select PPV-resistant sources (Decroocq et al., 2014). On as a signalling molecule (second messenger) that mediates
the other hand, genetic engineering constitutes a faster reli- responses to various environmental stresses (Neill et al.,
able approach for inducing resistance to PPV. In recent years, 2002). Moreover, reactive oxygen species (ROS) such as
PPV-resistant transgenic plants have been obtained using the H2O2 play a key role in signal transduction pathways in
expression of viral-derived sequences, including hairpin- order to adjust the cellular machinery to an altered condi-
containing viral transgenes (Di Nicola-Negri et al., 2005; tion (Jaspers and Kangasjrvi 2010; Miller etal., 2010).
Ilardi and Di Nicola-Negri, 2011; Di Nicola et al., 2014), In later works, the effect of long-term PPV infection on the
and single-chain variable fragments specific to the NIb RNA response of antioxidative metabolism at the subcellular level
PPV replicase (Gil et al., 2011). However, the regeneration was studied in peach plants (Prunus domestica cv. GF305) and
and transformation of Prunus species is still difficult and in apricot plants (Hernndez etal., 2004, 2006; Diaz-Vivancos
is limited to a few genotypes (Petri and Burgos, 2005; Petri etal., 2006). These authors observed that infection by PPV
etal., 2008). produced an imbalance in the antioxidative metabolism and
Changes in cell ultrastructure induced in described under both biotic and abiotic stress situations. An
PPV-infected plants. increase in the number of plastoglobules due to an enhanced
plastid lipid metabolism has been reported in response to
PPV infection caused an increased generation of ROS, imbal- oxidative stress and during senescence (Austin et al., 2006).
ance in antioxidative metabolism, and proteome changes, These authors described that plastoglobules form linkage
mainly in the chloroplast (Hernandez et al., 2004, 2006; groups that are attached to each other and remain continu-
Diaz-Vivancos et al., 2008; Clemente-Moreno et al., 2013). ous with the thylakoid membrane during oxidative stress
Moreover, the alteration of fluorescence parameters in PPV- and senescence. The increase in the number and size of plas-
infected plants is accompanied by modifications in the chlo- toglobules observed in PPV-infected plants could thus be
roplast ultrastructure. Almasi etal. (1996) described a slight related to the establishment of oxidative stress during viral
deformation in the chloroplasts of PPV-infected Nicotiana disease development (Hernandez etal., 2006; Diaz-Vivancos
benthamiana plants. Later, ultrastructure studies were car- et al., 2008; Clemente-Moreno et al., 2013). Similar effects
ried out in peach, apricot, and pea leaves. Cells from leaves have been reported in other plantvirus interactions, such
showing sharka symptoms had fewer chloroplasts than those as in Zucchini yellow mosaic virus (ZYMV)-infected pump-
observed in control plants, and these chloroplasts showed kin leaves, in which the amount of plastoglobules increased
a disorganized structure and dilated thylakoids, a reduced significantly, whereas the amount of thylakoids decreased
amount of grana and starch content, and an increase in the (Zechmann etal., 2003).
number and size of plastoglobules (Fig.5) (Hernandez etal.,
2006; Diaz-Vivancos et al., 2008; Clemente-Moreno et al.,
2013). The decrease in starch content in the chloroplast can be Proteome changes induced in
explained by different hypotheses. PPV-infected leaves could PPV-infectedplants
have increased demand for respiration, caused by activated
defence responses and by the requirements of the pathogen Changes in protein synthesis produced by virus infections
(Ayres et al., 1996). In addition, PPV infection reduced the have been known for a long time (Comacha and Sanger,
expression of some proteins related to carbohydrate metab- 1982). Information about the effect of PPV infection on the
olism, such as aldolase and ADP-glucose pyrophosphory- proteome of its hosts is very scarce. Diaz-Vivancos et al.
lase (Clemente-Moreno et al., 2013). Decreased aldolase (2006) published the first information about the effect of
expression induced a decrease in starch (Haake etal., 1999), PPV on differential apoplastic protein expression in woody
whereas ADP-glucose pyrophosphorylase is the major regu- plants. The identification of proteins using MALDI-TOF
lating enzyme in starch biosynthesis (Tiessen etal., 2002). (matrix-assisted laser desorption/ionization-time of flight)
The presence of dilated thylakoids and an increase in plas- and peptide mass fingerprinting analyses showed the induc-
toglobules (lipoprotein particles inside chloroplasts) seem tion of a thaumatin-like protein [a pathogenesis-related (PR)
to be a general stress response and have been previously protein] as well as a decrease in mandelonitrile lyase [MDL;
Page 6 of 11|Clemente-Moreno etal.
PPV-induced changes in gene expression susceptible to the virus (cv. Screara). These authors described
that fragments coding for myosin, kinesin, transketolase, and
Cloning genes that are up- or down-regulated by a viral infec- the ankyrin like-protein were overexpressed in Goldrich and
tion can provide new insights into susceptibility to PPV or associated with susceptibility. Both myosin and kinesin are
resistance mechanisms (Decroocq etal., 2005). Different can- involved in intracellular motile processes (Kinkema et al.,
didate genes have been described to be involved in the resist- 1994) and might play a role in the regulation of cell to cell
ant or susceptible response to PPV in plants. The eukaryotic transport. Transketolase takes part in both the Calvin cycle
translation initiation factor eIF4E and genes involved in RNA and the oxidative pentose phosphate pathway (OPPP), playing
silencing pathways are among the candidate genes of inter- an important role in photosynthesis and in phenylpropanoid
est identified. The eIF4E factor and its isoforms [eIF(iso)4E] metabolism (Henkes etal., 2001). These results agree with the
have been shown to control susceptibility or resistance to data observed in a susceptible pea cultivar. In this case, an
virus infection in many plant species (Robaglia and Caranta, increase in transketolase protein abundance was observed in
2006; Wang et al., 2013). However, in most cases, inherit- PPV-infected leaves at 15 d post-inoculation (Diaz-Vivancos
ance of the resistance associated with these factors has been et al., 2008). The OPPP, besides its important function in
shown to be recessive. On the other hand, products of the NADPH production, also supplies ribose-5-phosphate for the
RNA silencing pathway are known to have an implied role in synthesis of ribonucleotides, which can be required for virus
antiviral defence pathways (Yu etal., 2003; Vaucheret, 2008). replication in the cytosol (Diaz-Vivancos etal., 2008). Also, a
Escalettes etal., (2006) analysed the effect of PPV infection cDNA coding for a putative classIII chitinase was repressed
on the gene expression profile using cDNA-amplified frag- in infected plants from the resistant genotype and expressed
ment length ploymorphism (AFLP) in two apricot cultivars, in the susceptible cultivar Screara, indicating that chitinases
one partially resistant to PPV (cv. Goldrich) and the other can be putatively involved in the compatible interaction
Page 8 of 11|Clemente-Moreno etal.
(Escalettes etal., 2006). Plant chitinases are defence proteins also increased GSH levels and GSH-related enzymes in sev-
induced by environmental stress conditions (Collinge et al., eral plant species, such as sorghum, spinach, tobacco, pop-
1993). Moreover, PPV infection increased the expression of lar, pumpkin, pea, and peach (Hausladen and Kunert 1990;
NPR1 in peach GF305 under in vitro conditions (Clemente- Hilton etal., 1990; Gullner etal., 1999; Komives etal., 2003;
Moreno et al., 2012). Furthermore, the salicylic acid- (SA) Zechmann etal., 2007; Clemente-Moreno etal., 2010, 2012,
related induction or the overexpression of NPR1 leads to an 2013). These treatments were also reported to induce pro-
increase in the induction of PR gene expression and enhanced tection against different types of viruses, including TMV,
disease resistance (Cao etal., 1998; Kinkema etal.,2000). ZYMV, and PPV (Gullner etal., 1999; Zechmann etal., 2007;
Microarray analysis of PPV-infected Arabidopsis plants Clemente-Moreno etal., 2010). BTH is a functional analogue
showed the induction of genes involved in soluble sugar, of SA and has also been demonstrated to provide protec-
starch, and amino acid metabolism; intracellular membrane/ tion against different pathogens, including fungi, bacteria,
membrane-bound organelles; chloroplasts; and protein fate. and plant viruses (Friedrich etal., 1996; Grlach etal., 1996;
On the other hand, genes related to development/storage pro- Lawton etal., 1996; Anfoka, 2000). However, studies on the
teins, protein synthesis and translation, and cell wall-associ- use of OTC or BTH in PPV-infected plants are limited to
ated components were down-regulated (Babu et al., 2008). three papers published by our group based on different mod-
More recently, Pagny etal. (2012) reported the identification els: herbaceous plants (pea) and woody plants (peach), under
of genomic regions from A.thaliana associated with suscep- both in vitro and greenhouse conditions (Clemente-Moreno
tibility to PPV, in particular to the long-distance movement etal., 2010, 2012, 2013). In PPV-infected plants, both OTC
of the virus, such as RTM3-like TRAF domain-containing and BTH treatments reduced the severity of sharka, meas-
genes and MATH and SHA3genes. ured as a percentage of leaves showing symptoms. In pea
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of the antioxidant enzymes was different in PPV-compatible enzymes. The Plant Cell 18, 16931703.
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