Grapevine leafroll (GLD) and Rugose wood (RW) are two diseases of great concern for grapevine industries worldwide. GLD causes degeneration of phloem cells, preventing the translocation of synthesised carbohydrates from the leaves. The disease delays maturation and decreases the sugar content of berries and ultimately, negatively influences the quality of wine produced. In RW-affected plants, abnormal activity of cambium cells affects graft take of cultivars to rootstocks. This leads to reduced vigour of grapevines, and ultimately result in lower productivity and longevity of vineyards.
Grapevine leafroll (Photo: Hanno van Schalkwyk).
Rugose wood (Photo: Piet Goussard).
Investigations revealed the association of viruses of the families Closteroviridae and Betaflexiviridae with GLD and RW disease, respectively. These conclusions were based on the consistent association of members of these families in disease-affected grapevines, mostly grapevine leafroll associated virus 3 (GLRaV-3) in GLD-affected plants and grapevine virus A (GVA), and grapevine virus B (GVB) respectively in Kober stem grooving (KSG) and corky bark (CB), two diseases of the RW complex. Although the author agrees with the common held belief that these viruses are the probable cause of the diseases, unequivocal proof, such as the fulfilment of Koch’s third postulate, is still required. To fulfil Koch’s third postulate, isolation of a virus and re-infection of the host, with concomitant development of disease symptoms, is required. Grapevines are usually infected with multiple viruses often with the same insect vectors, and separating them in the host is practically impossible. While some of viruses, for example GVA and GVB, have successfully been transmitted by mechanical transmission or insect vectors and isolated from alternative herbaceous hosts, transmission back to grapevines remains elusive. It has been shown that the genome of grapevine leafroll associated virus 2 (GLRaV-2) changes to adapt to its herbaceous host, which renders it unable to re-infect grapevine (Kurth et al., 2012). Therefore to obtain a pure inoculum of grapevine viruses associated with GLD and RW that is not mechanically transmissible, and to avoid using experimental herbaceous hosts, biologically active cDNA copies (“infectious clones”) of these viruses can be constructed.
The technique used to construct infectious cDNA clones of plant viruses has been known since 1984 (Ahlquist et al., 1984). Technology has since improved and is well established in scientific literature. In short, the construction of an infectious clone requires the conversion of the virus RNA genome to DNA and inserting the DNA into a vector that contains all the required controller elements. Typically, these vectors can then be delivered to grapevine cells using a non-pathogenic laboratory strain of Agrobacterium tumefaciens. Although the technology has been available for more than 30 years, it has not been widely used for grapevine viruses. The first grapevine cDNA clones were constructed for GVA and GVB, about 17 years ago (Galiakparov et al., 1999; Sardarelli et al., 2000), however, infection of grapevines using a cDNA clone has not been achieved until recently with grapevine leafroll associated virus 2 (GLRaV-2) (Kurth et al., 2012). Despite the advancements in this technology, it remains challenging to construct cDNA clones that are biologically active and stable. An additional challenge, especially crucial for grapevine, is the relatively low frequency of cDNA construct delivery to grapevine cells. This relatively low frequency of infections is not a problem when investigating the aetiology of grapevine diseases with suspected viral aetiology, such as GLD and RW, as even finding only a few virus-infected plants would successfully demonstrate this. The challenge is in obtaining a large number of grapevine cells with actively replicating viruses that move from cell to cell and then systemically infect the plant.
The infection of grapevine cells with a virus using its cDNA clone still does not guarantee the success of the experiment. The replicating virus is the target of the plant’s anti-virus defence system called RNA interference (silencing), which is another important challenge in grapevine virus aetiological studies. While most grapevine viruses encode proteins that suppress silencing by the host, indications are that the suppressor activity of proteins encoded by GVA, and possibly by other members of the family Betaflexiviridae, is relatively weak in contrast to members of the Closteroviridae family, like GLRaV-3, (Chiba et al., 2006). This may be the reason that grapevines affected by KSG and CB are frequently infected with GVA and GVB respectively along with a member of the Closteroviridae family, usually GLRaV-3 in South Africa. Thus, even if infectious and stable cDNA clones of GVA and GVB, are constructed they might need protection from silencing by viruses encoding stronger suppressors. This further complicates the study of RW disease aetiology. While the possible importance of suppression of gene silencing in the development of GLD and RW diseases is based on current knowledge, there may be other undiscovered factors hampering the progress of virus infection after agro inoculation of grapevines with cDNA clones of viruses. Among the papers mentioned earlier on construction of cDNA clones of members of the family Betaflexiviridae, GVA, GVB (Galiakparov et al., 1999; Sardarelli et al., 2000) and also recently, grapevine rupestris stem pitting associated virus (GRSPaV) (Meng et al., 2012), there is no convincing evidence on the spread of these viruses in grapevines. Furthermore, while infectious cDNA clones of two members of the family Closteroviridae, GLRaV-3 (Jarugula et al., 2010) and GLRaV-2 (Kurth et al., 2012), were recently developed, it was only GLRaV-2 that clearly moved systemically in grapevines.
In our laboratory, cDNA clones of each of the genetic variants of GVA and GVB associated with Shiraz disease (SD) and CB, were constructed (Goszczynski, 2015). Although clones of both of these viruses from N. benthamiana are biologically active, infection of grapevines was only successful for GVA. The reason for the lack of biological activity for the GVB cDNA clone in grapevines is not known. The cDNA clone of GVA was successfully agro infiltrated to in vitro propagated grapevine (cv. Shiraz) plantlets, where the virus spread and was easily detected by RT-PCR in leaf petioles of three consecutive new growths over about 8,5 months after agro infiltration. Thereafter the titres dropped significantly to almost undetectable levels.
Having a stable cDNA clone of a virus, which can be used to efficiently infect grapevines and replicates to a high titre and moves systemically, offers many opportunities for the study of the interaction between virus and plant in the development of a disease. Because of the dsDNA nature of clones, parts of the genome of a virus can be easily modified, and the modification can be tested in plants. Once the involvement of GVA and GVB in the aetiology of SD and CB respectively have been confirmed, these clones could be used to determine which genome part of these viruses is responsible for de-regulation of grapevine tissue development that leads to the symptoms of swollen and non-maturing canes of affected plants in both diseases.
It is commonly accepted that Grapevine leafroll and Rugose wood diseases are caused by viruses of the families Closteroviridae and Betaflexiviridae. Although the association has been shown, Koch’s third postulate is yet to be fulfilled, i.e. isolation and re-infection of the pathogen resulting in the development of disease symptoms. This is especially challenging for grapevine since they are mostly not mechanically transmissible. An alternative approach is to construct biologically active complementary DNA (cDNA) clones (“infectious clones”) of these viruses. The application of this technique to study the aetiology of grapevine diseases is, however, rather limited due to its technical difficulty. The main reasons are the lack of full length infectious clones for most grapevine viruses, the low success rate of delivery of cDNA clones to grapevine, and the weak suppressor activity of some viruses (GVA, GVB and GRSPaV).
The author would like to thank Winetech (Project No 11729) and the National Research Foundation (NRF) (Grant No 76699) for financial assistance towards this research. Opinions expressed and conclusions arrived at are those of the author and are not necessarily attributed to the NRF.
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– For more information, contact Dariusz Goszczynski at email@example.com.