Construction of cDNA clones of the genetic variants of grapevine virus A and grapevine virus B associated with Shiraz disease and corky bark disease to confirm the aetiologies of these diseases
Goszczynski, D E
ARC Plant Protection Research Institute
Despite the observed association of grapevine virus A (GVA) and grapevine virus B (GVB) with Shiraz disease (SD) and corky bark disease (CB), respectively, there is still no firm scientific proof that these viruses are the cause of these destructive diseases. Transmission of pure cultures of GVA and GVB back to grapevines, which must be followed by the development of SD and CB symptoms (fulfilment of the third Koch’s postulate), is necessary to confirm the virus aetiology of these diseases. With the current knowledge, it can only be achieved by infecting grapevines with GVA and GVB using cDNA copies of viruses. In this study we constructed full cDNA clones of two genetic variants of GVA and GVB associated with SD and CB and tested their pathogenicity to Nicotiana benthamiana, an alternative host of these viruses, and grapevines.
A standard approach was applied to construct cDNA clones of GVA and GVB. The virus genomes were RT-PCR amplified from dsRNA isolated from virus-infected plants in four overlapping DNA fragments containing unique restriction sides in overlapping regions. The amplicons were cloned to pGEM-T Easy vector (Promega), and then assembled to DNA complementary to the full genomes of these viruses in the same vector. The DNA clones of the viruses, under cauliflower mosaic virus (CaMV) 35S promoter, were re-cloned to various binary plasmids (pCAMBIA 1302 and modified pCB302) and then transformed to Agrobacterium tumefaciens strain C58C1 and agro inoculated to N. benthamiana to test the biological activity of the constructs. This general strategy of constructing DNA clones of viruses was first tested using GVA and GVB multiplied in Nicotiana benthamiana, and then applied to construct cDNA clones of these viruses from grapevines. A biologically viable cDNA clone of GVA from grapevine (isolate M5v) was vacuumagroinfiltrated to in vitro-propagated plantlets of Shiraz clone 22B. After a few months of plant growth, dsRNA was isolated from leaf petioles and tested for the presence of GVA using virus specific RT-PCR. RT-PCR and RT- nested PCR for specific amplification of GVA and members of the Foveavirus and Vitivirus genera, respectively, and cloning of PCR amplicons, single strand conformation polymorphism (SSCP) analysis of clones and sequencing were used to monitor the presence of GVA M5v in agroinfiltrated plants at different times of their growth. These techniques were also applied to re-investigate the virus status of SD-free mother Shiraz 22B and siblings plants used in the study. All SD-free and SD-affected plants used in agro infiltrations were also investigated for the presence of GLRaV-3 using virus-specific RT-PCR and RT- nested PCR based on degenerate primers specific to members of the family Closteroviridae.
In 2014, before this project officially started, cDNA clones of GVA and GVB, isolates M5-1 and 953-1 respectively, were constructed using dsRNA isolated from the alternative herbaceous host of these viruses, Nicotiana benthamiana. The clones induced systemic symptoms in this host similar to symptoms induced by the wild type of these viruses. The results are included in this report. In this study, a biologically viable and stable cDNA clone of GVA isolate M5v was constructed using dsRNA isolated from Shiraz disease (SD)-affected grapevine cv. Merlot. The genome of the variant shares 99.8 % nt identity with the variant M5-1 maintained in N.benthamiana. The cDNA clone of GVA M5v was successfully agro infiltrated to 8 in vitro propagated plantlets of Shiraz. One of these plants was pruned 3 times to investigate the presence of the M5v variant in new growths. The results of RT-PCR revealed that the virus could only be easily amplified from dsRNA isolated from this plant up to about 8 months after agro infiltration. Later its titre dropped significantly. The virus could not be detected serologically using Western Blot technique. Two years after agro infiltration, 3 of 8 plants successfully infected with GVA M5v using cDNA clone of this variant exhibited clear cane symptoms of SD. Once again, the virus could be easily amplified using RT-PCR. The M5v identity of GVA infecting these plants was confirmed by cloning and sequencing of RT-PCR amplicons. The extensive investigation of positively agro infiltrated grapevines for the presence of GVA M5v lead to the unexpected discovery that the mother Shiraz clone 22B and in vitro propagated sibling plants used in agro infiltrations with cDNA clone of M5v were infected with populations of various genetic variants of GRSPaV, GVA and GVB. These “contaminating” viruses” were present in Shiraz 22B grapevines in a consistently very low titre and hardly detected by standard two step RT-PCR. The investigation also revealed that the mother Shiraz 22B and sibling plants were also infected with GLRaV-3. The virus was hardly detected in the mother plant but its titre increased in sibling plants propagated in vitro. The virus was also present in SD-affected plants obtained in this study. Until now genetic variants of GLRaV-3 of groups I and II, and I, VI and VII were detected in the mother Shiraz 22B and SD-affected plants, respectively. Two attempts to construct a biologically active cDNA clone of GVB using dsRNA isolated from grapevines were not successful. Despite the fact that a full cDNA copy of this virus was assembled under Ca35S promoter, the construct was not infectious to N. benthamiana, and was not further investigated because of time constrain.
The results obtained in this study support hypothesis that GVA M5v is the key virus in inducing SD. The research data suggests that this genetic variant went to “latent stage” 8 months after the successful infection and induced the disease 15 months later. We discovered that the mother plant Shiraz 22B used in the study was infected with GRSPaV, GVA, GVB and GLRaV-3. The viruses were present in very low titre in this plant and were hardly detected using a standard two step RT-PCR technique. Although the origin of the infections is not known, extensive molecular heterogeneity of these viruses suggest the possibility that the viruses were not eliminated from the Shiraz mother grapevine when clone 22B was established. The methods of virus elimination used by the industry should be reevaluated using recently developed techniques of virus detection.