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Molecular Hybridization Techniques for Detecting and Studying Fruit Tree

Viruses and Viroids

Chapter · January 2011

DOI: 10.1094/9780890545010.059

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Molecular Hybridization Techniques for
Detecting and Studying Fruit Tree Viruses and
Viroids

V. Pallas, F. Faggioli, F. Aparico and J. A. Sanchez-Navarro

 INTRODUCTION

Fruit trees are affected by a large number of different and economically

important viruses and viroids. Once a virus or viroid infects a plant, the progress of
infection cannot be prevented or controlled. Therefore, to slow down the spread of
viruses in the field, it is important to detect these viruses in their early stage of infection
and eliminate the infected plants. Recently, diagnostic methods have been developed to
improve the sensitivity and reduce the processing time and cost. Thus, efforts have been
directed to achieve the simultaneous detection of several viruses in a single assay. Many
of these techniques are PCR-based with the associated problem of false positive results.
These may be due to contamination or non-specific amplification. Steps to reduce the
incidents of false positive results increase the value of these techniques for routine
diagnosis (e.g. James et al., 2006; Lopez et al., 2008). In spite of the fact that molecular
hybridization is a less common methodology in diagnostic laboratories, this technique
offers great advantages when compared to PCR-based methods. For example, molecular
hybridization allows for near-total absence of contamination problems and a much
greater flexibility. Indeed, its specificity can be adjusted to make the hybridization test
as specific or as general as required.
The potential of molecular hybridization as a diagnostic tool in Plant Virology
was first demonstrated for the detection of viroids (Owens and Diener, 1981) for which
no serological method could be used due to the lack of protein component in their
structural constituents. The technique was later applied to plant viruses (Maule et al.,
1983; Garger et al., 1983). The basic principles of molecular hybridization are beyond
the scope of this chapter and will only be briefly discussed here. Several aspects
affecting the different steps of the molecular hybridization technique (which include the
synthesis of the labeled probe, sample preparation, hybridization and detection) have
been described in previous reviews (e.g. Hull, 1993; Pallás et al., 1998; Mühlbach et al.,
2003).
Molecular hybridization is based on the specific interaction between
complementary purine and pyrimidine bases forming A-T and G-C base pairs, which
results in a stable hybrid formed by part (or the totality) of the nucleic acid sequence of
the pathogen to be detected (target molecule) and by the labeled complementary
sequence (probe). The stability of the hybrid depends upon the number of hydrogen
bonds formed, and upon both electrostatic and hydrophobic forces. Electrostatic forces
rely on the phosphate molecules of the nucleic acid backbone, whereas hydrophobic
interactions are maintained between the staggered bases.
Different molecular hybridization approaches have been developed, all of them
based on the same principles but differing in the way the target molecule is applied and
immobilized onto the solid support. Here we review the different formats of the
molecular hybridization methodology and describe the main applications on fruit tree
viruses and viroids: dot-blot hybridization, tissue-print, multiplex hybridization, in situ
hybridization and microarrays detection.

DOT-BLOT HYBRIDIZATION

The most common molecular hybridization format is dot-blot hybridization. This

involves the direct application of a nucleic acid solution onto a solid support, such as
nitrocellulose or nylon membranes, and the subsequent detection with appropriate
specific probes (Fig.1). The use of non-radioactive precursors to label nucleic acids has
made the molecular hybridization technique more accessible. The first non-isotopic
probes used for routine diagnosis of viroids were developed by biotinylation with
photobiotin of plasmid vectors containing full-length monomer viroid inserts.
Photobiotin-labeled DNA probes were developed for diagnosis of Avocado sunblotch
viroid (ASBVd), Coconut cadang-cadang viroid (CCCVd), Chrysanthemum stunt
viroid (CSVd) and Potato spindle tuber viroid (PSTVd) (McInnes et al., 1989). The
sensitivities obtained were similar to those obtained with radioactive probes. The
biotinyl labeled nucleic acids are recognized with great efficiency by avidin or its
microbial analogue, streptavidin, taking advantage of the exceptionally high affinity of
the avidin-biotin complex. The main disadvantage of this system is that when sap
extracts are used, the endogenous biotin may cause false positives or, alternatively, the
presence of glycoproteins that bind avidin or biotin-binding proteins can give high
background. Thus, digoxiygenin (DIG) became the most widely used non-radioactive
precursor for labeling nucleic acids. This molecule is bound via a spacer arm (11-carbon
residues) to uridin nucleotides and is incorporated enzymatically into nucleic acids by
standard methods. Non-radioactive DIG-labeled probes were first applied to detection
of viroids (e.g. Roy et al., 1989; Candresse et al. 1990; Podleckis et al. 1994) and later
to plant viruses (Más et al. 1993).
 The majority of plant viruses and viroids have RNA genomes and RNA–RNA
hybrids are more stable than RNA–DNA hybrids. Therefore, more stringent
hybridization conditions can be selected in the case of RNA–RNA hybrids, allowing the
increasing of specificity and reducing the non-specific background signals. Thus,
greater preference is given to RNA probes than to DNA probes for pathogen detection.
Non-radioactive RNA probes (riboprobes) are synthesized by incorporating DIG into
cRNA by means of an in vitro transcription reaction from cloned viral cDNA. Nucleic
acid targets are fixed to the membrane by baking at 80C for 2 h or at 120C for 30 min,
or by UV cross-linking (in the last two cases, positively charged nylon membranes are
preferred). The latter method results in a 5-10 fold increase over the baking methods.
Hybridization depends on several factors such as the complexity (length and
composition of the nucleic acid) and concentration of the probe, temperature, salt
concentration, base mismatches, and inclusion of hybridization accelerators. The
temperature at which half the strands are disassociated is the melting temperature (Tm).
The stringency of the hybridization conditions and the stability of the hybrid complexes
determine the specificity of hybrid formation. In general, higher temperatures and low
salt concentration increase stringency. Apart from increasing stringency, the presence of
formamide in the hybridization solution not only favors correct base pairing, but also
decreases the background and avoids riboprobe degradation. For plant RNA-virus
detection, hybridizations are often carried out at 65–68C, while with viroids, good
signal/background ratio is achieved at 70–72C in 50% formamide. Hybridized filters
may be processed immediately or stored dry for processing later. The labeled hybrids
are detected by an ELISA-like reaction, using high-affinity DIG-specific antibodies
conjugated to alkaline phosphatase. After three washing steps, a detection reaction is
obtained by subsequent addition of alkaline phosphatase substrates, either the color
substrates BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (nitroblue
tetrazolium) or the chemiluminescent substrate CSPD® (disodium 3-(4-
methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro) tricyclo [3.3.1.13,7] decan}-4-yl) phenyl
phosphate; Roche Molecular Biochemicals, Mannheim, Germany). When clarified sap
is blotted on the membrane instead of a more purified sample, the colorigenic detection
method can produce excessive background due to the ability of interfering substances
remaining in such blots to chemically reduce the substrate, but light emission is not
affected (Más et al. 1993).
 Radioactive dot-blot hybridization was first developed to detect Plum pox virus
(Varveri et al., 1988; Wetzel et al., 1990) which was later modified to utilize
digoxinenin-labeled riboprobes (e.g Herranz et al., 2005). Dot-blot hybridization using
radiolabeled probes allows the discrimination between healthy and virus-infected
material throughout the growing season (Scott et al., 1992) and is suitable for detecting
Prunus necrotic ringspot virus (PNRSV) serotypes that react poorly in ELISA (Crosslin
et al., 1992). Powell et al. (1991) developed a cDNA riboprobe for the detection of
Tomato ringspot virus (ToRSV) in nectarine trees. Nevertheless, as stated above, the
use of radiolabeling renders this approach unsuitable for routine diagnosis. Non-isotopic
RNA probes have been used to monitor PNRSV infection after in vitro micrografting
(Heuss-La Rosa et al., 1995) and to detect this virus in field conditions (Sánchez-
Navarro et al., 1998; Pallás et al., 1998; Saade et al., 2000; Herranz et al., 2005). Non-
isotopic dot-blot hybridization has also been applied to detect Apple chlorotic leaf spot
virus (ACLSV) (Dominguez et al., 1998), Cherry mottle leaf virus (CMLV) (James et
al. 1999) and American plum line pattern virus (APLPV) (Alayasa et al., 2003; Al
Rwahnih et al., 2004).
The use of cRNA riboprobes using either radioactive or non-radioactive
molecules for the labeling has been the method of choice for detecting known viroid
sequences and has been applied to a large number of viroids (Potato spindle tuber
viroid and/or Apple scar skin viroid (ASSVd), Hadidi et al., 1990; Podleckis et al.,
1993; Kyriakopoulpo et al., 2001; Citrus exocortis viroid (CEVd) and Avocado sun
blotch viroid (ASBVd), Romero-Durban et al., 1995; Pear blister canker viroid
(PBCVd) and Peach latent mosaic viroid, Ambros et al, 1995; Loreti et al 1995;
Kyriakopoulou et al., 2001; Michelluti et al., 2005; Hop stunt viroid, Amari et al., 2001
a,b ; Astruc et al., 1996; Citrus viroids, Palacio et al., 2000). Viroid RNA extraction can
be done using organic or non-organic solvents, special matrices and/or other reagents or
procedures able to separate the nucleic acids from the other cell components. The
obtained target, depending on the extraction method, would be composed of either total
nucleic acids (TNAs), total RNAs (TRNAs) or the purified viroid RNA present in the
viroid-infected plant tissues. In recent years, the use of phenol or other toxic organic
solvents is decreasing and methods that avoid their use have been developed
successfully (Astruc et al., 1996; Cañizares et al., 1998). Commercial kits for total
RNAs extraction are also available but their use is more suitable for RT-PCR detection
than for molecular hybridization (F. Faggioli, unpublished data).
 TISSUE-PRINT HYBRIDIZATION

For routine analysis, sample manipulation must to be reduced to a minimum.

This can be easily achieved by using the tissue-printing technique that avoids the
sample extraction and only requires the direct transfer of the plant material (stem,
cutting, leaf, fruit) to a nylon or nitrocellulose membrane (Fig.2). This technique was
first described to detect proteins by immunocytolocalization (Cassab and Varner, 1987)
and was later used for RNA detection (McClure and Guilfoyle, 1989). The same
approach was then adapted for detection and localization of plant viruses (Mansky et al.,
1990; Chia et al., 1992; Más and Pallás, 1995) and viroids (e.g. Podleckis et al., 1993;
Romero-Durbán et al., 1995; Astruc et al., 1996; Hooftman et al., 1996; Hurtt et al.,
1996; Palacio et al., 2000; Torres et al., 2004; Mandic et al., 2008). The imprint
hybridization technique can be applied not only for diagnostic purposes with the
obvious advantage of reducing the test times (see previous references) but also to study
virus/viroid distribution within the infected plant (e.g. Más and Pallas, 1996; Stark-
Lorenzen et al., 1997; Amari et al. 2007a,b).
Tissue-imprinting hybridization, has been applied in field conditions for large
scale indexing of the presence of HSVd in apricot trees (Astruc et al., 1996; Cañizares
et al., 2001), PLMVd in nectarine and peach trees (Loreti et al., 1999; Torres et al.,
2004) or for rapid detection of ASSVd in pear (Hurtt et al., 1996), to monitor the
infection of HSVd in apricot trees (Amari et al., 2001a) over a whole growing season, to
study the etiology of the apricot ‘viruela’ disease (Cañizares et al., 2001) or to track
PNRSV, ApMV and PDV in stone fruit trees throughout the year (Matic et al., 2008).

MULTIPLEX DETECTION

To save time, reduce labor and overall costs, non-isotopic molecular

hybridization techniques were developed for simultaneous use of up to six riboprobes.
Initially developed for phytosanitary certification of tomato (Saldarelli et al. 1996) and
simultaneous detection of five viruses affecting carnation (Sánchez-Navarro et al. 1999)
it was applied to ilarviruses affecting stone fruit trees (Saade et al. 2000). In all cases,
the mixture of corresponding riboprobes did not significantly reduce the sensitivity
when compared to detection with individual riboprobes. In some instances, mixtures of
many riboprobes resulted in undesirable background that can make the results
indecipherable. This may be overcome by treating the membranes with RNase A after
the washing steps, which then results in clear specific hybridizations (Sánchez-Navarro
et al. 1999). A recent alternative to the mixture of different riboprobes is the use of
polyprobes (Herranz et al. 2005). This strategy involves the cloning, in tandem, of
partial nucleic acid sequences of different viruses so as to allow for the synthesis of a
unique riboprobe called polyprobe. Polyprobes have been developed to detect two, four,
or six stone fruit or tomato viruses (Herranz et al., 2005; Aparicio et al., 2008) and for
detecting four viroids affecting citrus trees (Cohen et al., 2006) or a combination of six
viruses plus two viroids affecting stone fruits crops (Sanchez-Navarro et al., manuscript
in preparation). The polyprobes were found to be comparable to the individual
riboprobes in terms of limit of detection, and specificity. In addition, the position in
which a specific probe is located in the polyprobe does not affect the sensitivity. The
validity of this new simultaneous detection strategy was confirmed by the analysis of 46
field samples that included up to seven different hosts from 10 different geographical
regions (Herranz et al. 2005). The polyprobe strategy offers several obvious advantages:
(i) only one synthesis reaction is needed to detect up to six viruses, thus saving time and
costs, and avoiding potential variability in the efficiency of the different riboprobes; (ii)
unlike the mixture of riboprobes, this strategy does not require the RNase A treatment to
avoid the undesirable background observed when high amounts of DIG–RNA probes
are present in the hybridization solution, thus allowing the detection of both DNA and
RNA viruses in the same assay; and (iii) the polyprobe concept may allow for the
design of a specific “crop probe” to detect a number of different viruses, and possibly
other pathogens affecting that crop.

IN SITU HYBRIDIZATION

This format of molecular hybridization is the method of choice to study the

subcellular location of fruit viruses and viroids within the plant cell or to address the
translocation and final distribution of the pathogen through the plant (Fig.3). Paraffin-
embedded tissue is the most commonly used material for these purposes. Harders et al.
(1989) used in situ hybridization to demonstrate that PSTVd accumulates in the nuclei
of infected cells. Detection of ASBVd in the thylakoid membranes (Bonfiglioli et al.,
1996) of avocado leaf chloroplasts has been made by in situ hybridizaion. Localization
of intermediates in the replication of ASBVd in chloroplasts suggests a site for in vivo
synthesis (Navarro et al., 1999). PLMVd also accumulates in the chloroplasts (Bussiere
et al., 1999). More recently Rodio et al. (2007) have used this approach to demonstrate
that PLMVd can invade the shoot apical meristem and induce alterations in proplastids,
bypassing the RNA surveillance system that restricts the entry of a nucleus-replicating
viroid and most RNA viruses.
For fruit viruses, in situ hybridization has been used to study the localization of
Plum pox virus (PPV) RNA in stems of susceptible and resistant apricot cultivars (Ion-
Nagy et al., 2004), to study the plant distribution of Cherry leaf roll virus (CLRV) (Más
et al., 2000), to demonstrate the presence of PNRSV within pollen grains of nectarines
(Aparicio et al.,1999) and to study the route of infection and the pattern of distribution
of PNRSV in apricot pollen grains and tubes (Amari et al., 2007a) revealing that
PNRSV distribution follows the same pattern as the cellular components required for
pollen tube germination and cell wall tube synthesis.

DNA MICROARRAY TECHNOLOGY

In recent years, DNA microarray technology greatly extended the power of

nucleic acid hybridization. The microarray technology was first reported by Schena et
al. (1995) for gene expression analysis. It works as a reverse hybridization method,
where thousands of probes are linked onto a solid support, generally a microscope glass
slide (because glass has low intrinsic fluorescence and is chemically inert) covered by a
positively charged poly L-lysine film. Initially, the probes consisted of PCR products
(200-1000 bp) amplified from cDNA libraries or genomic DNA (Schena et al., 1995).
However, this method is laborious, expensive and time consuming. An alternative
approach was developed which involved the use, as probes, of synthetic cDNA
oligonucleotides typically ranging from 20 to 70 nucleotides. The oligonucleotides can
easily be designed and modified by adding some particular reactive groups to better
bind them onto the surface of the slide or to orientate the binding. The optimal capture
probe length depends upon the practical application. Generally, 70 nucleotides have
been documented to give high performance results (Hughes et al., 2001; Wang et al.,
2002).
Several methods have been developed to spot the slides. The most widely used
method involves the use of a robot that spots, onto the slides, the oligonucleotide
sequences previously synthesized and whose specific positions had previously been
recorded by the software. The probes are picked up by individual pins through capillary
action and are deposited onto the surface by using tension forces existing between the
spotting buffer and the surface (Auburn et al., 2005; Schena et al., 1995). The spotting
of the probes is also possible using non-contact methods like thermal or piezoelectric
inkjet printing (Agilent, Palo Alto CA).
The nucleic acid target (usually RNA) can be reverse-transcribed as cDNA or
amplified (dsDNA) and then labeled before hybridization. The labeling of the targets is
usually done using two different dyes: Cyanine 3 (Cy3) and Cyanine 5 (Cy5). The target
can be labeled using a direct or indirect labeling reaction. In the direct reaction, a Cy3-
or Cy5-fluorescently labeled dCTP is incorporated during the reverse transcription
reaction or during the PCR reaction (Lockart et al., 1996). In the indirect labeling, the
reverse transcription or the amplification reaction are performed using a chemically
reactive nucleotide (amino allyl-dUTP) which, during a second step, is bonded to the
reactive forms Cy3- or Cy5-NHS esters (Cox et al., 2004; Xiang 2002). In order to
detect the reaction, a high resolution confocal laser scanner is needed. The resulting
image shows each spot in different colors; red, for the probes reacting with Cy5-labeled
target, green, for the probes reacting with Cy3- labeled target, black for the probes
which do not react to any targets and yellow for the probes reacting to both dyes. The
image is then analyzed using software that transforms the intensity of light of each spot
to a value reflecting the ratio of red to green intensity (Hadidi et al. 2004).
DNA microarray hybridization analysis stands out for its simplicity,
comprehensiveness, data consistency, sensitivity and high throughput. It has the ability
to simultaneously display the expression of thousands of genes at a time. Thus, it is a
powerful tool for genetic analysis, including single nucleotide polymorphism typing.
DNA microarray methods have already made a marked impact on many fields in
biology and their potential use for the detection of viroids and other plant pathogens has
been recently discussed (Hadidi et al., 2004). Oligonucleotide microarrays are
particularly useful in the detection of pathogens because of the uniformity in probe
length, which significantly reduces the chances of cross-hybridization and allows
distinguishing between single base mismatches. Both short and long oligonucleotide-
based approaches have been described for the simultaneous detection and identification
of many pathogens in a DNA microarray hybridization analysis (Wilson et al., 2002;
Wang et al., 2002).
 Several studies were published in 2003 regarding the microarray based detection
of some potato viruses. In these reports, the microchip consisted of arrays of spotted
PCR products (Lee et al., 2003; Bystrika et al. 2003). In 2005, microarray chips, spotted
with synthetic oligonucleotides of 40 nt, were used to detect five viruses affecting
potatoes (Bystricka et al.2005). Abdullahi et al. (2005) and Du et al. (2007)
demonstrated the potential of the microarray technique when detecting PSTVd from
potato field samples. The sensitivity of this test was the same as that of dot-blot
hybridization performed on nylon membrane. More recently, a microarray chip spotted
with synthetic nucleotides of 70 nt in length, has been developed in order to detect and
genotype different Plum pox virus (PPV) strains in PPV-infected peach, apricot and N.
benthamiana leaves (Pasquini et al., 2008).

CONCLUSIONS AND FUTURE PERSPECTIVES

Molecular hybridization approaches in the field of phytodiagnosis already appeared to

be a very promising tool in the early eighties, especially in viroid research. However,
this methodology required a minimal level of experience in basic molecular biology and
the availability of specialized equipment which was lacking in non-specialized
laboratories. With the emergence of PCR-based methods in the early nineties which
avoided all these requirements and rendered a higher sensitivity, molecular
amplification assays became more popular in non-specialized laboratories.
Nevertheless, the lower sensitivity of the hybridization techniques is compensated by a
higher reliability (absence of contamination and false positives) and a better versatility
than RT-PCR. Moreover, the development of protocols that avoids the use of dangerous
reagents, such as radioactive labels or organic solvents and the possibility to detect
multiple pathogens in a single assay (e.g. polyprobes), has made molecular
hybridization a more useful and versatile technique for the study of fruit tree viruses and
viroids. The main restriction to this methodology is probably the production of the
probes themselves. However, with the future prospect of labeled-probe synthesis carried
out directly in a simple PCR event (dsDNA probe) or of commercialization of
digoxigenin-labeled riboprobes, the use of the molecular hybridization techniques could
be made easier and more accessible even to non-specialized laboratories. Moreover, the
DNA microarray technique opens up new perspectives in the field of molecular
hybridization due to its enormous potential. In fact, not only is it versatile, but it also
greatly expands the spectrum of detectable viruses/viroids in a single assay, while
simultaneously enabling discrimination among the detected viruses/viroids.

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