Differential pathogenicity of two different recombinant PVYNTN isolates in Physalis floridana is likely determined by the coat protein gene
© Hu et al; licensee BioMed Central Ltd. 2011
Received: 22 November 2010
Accepted: 7 May 2011
Published: 7 May 2011
A previous study has identified two types of recombinant variants of Potato virus Y strain NTN (PVYNTN) in China and sequenced the complete genome of the variant PVYNTN-HN2. In this study, the complete genome of isolate PVYNTN-HN1 was fully sequenced and analyzed. The most striking difference between the two variants was the location of recombinant joint three (RJ3). In PVYNTN-HN1, like other typical European-PVYNTN isolates such as PVYNTN-Hun, the RJ3 was located at nucleotide (nt) 9183, namely the 3' proximal end of the CP gene (nt. 8571-9371), thus leading to most (the first 613 nucleotides from the 5' proximal end) of the CP gene (801 bp) with a PVYN origin and PVYN-serotype; whereas in contrast, the RJ3 in PVYNTN-HN2 was located at nt 8572, consequently leading to a CP gene of PVYO origin and PVYO-serotype. The varied genome composition among PVYO, PVYN, PVYN:O, PVYNTN-HN1 and PVYNTN-HN2 made them useful for the investigation of possible roles of gene segment(s) in symptom formation on host plants. When Physalis floridana plants were infected with different PVY isolates, two types of symptoms were induced. PVYN and PVYNTN-HN1 induced mild symptoms (mainly mild mottling) whereas PVYO, PVYN:O and PVYNTN-HN2 induced serve symptoms including leaf and stem necrosis, leaf-drop and stunting. These results, together with a previous study using artificial PVY chimeras, demonstrate that the CP gene, especially the 5' proximal segment (nt 8572-9183), and/or CP likely determine the pathogenicity of PVY in P. floridana.
Potato virus Y (PVY) is the type species of the Potyvirus genus in the Potyviridae family . It infects a number of plant species in the nightshade family (Solanaceae) and causes a wide range of symptoms from symptomless to mosaic, mottling, lesions, stunting, necrosis and plant death, depending on the plant species, the cultivar, the virus strain and isolate . PVY possesses a single-stranded positive RNA genome comprised of approximately 9700 nucleotides that encode a polyprotein of approximately 3061 amino acids . The polyprotein undergoes proteolysis to form 10 mature proteins with different functions including replication, transportation and spread of the virus [1, 2]. Many strains/substrains of PVY have been recognized according to the primary hosts and host reactions. For the potato-infecting PVY, the ordinary strain (PVYO), the tobacco veinal necrosis strain (PVYN) and the potato stipple streak strain (PVYC) are the first ones to be recognized , followed by the potato tuber necrosis strain (PVYNTN) and the recombinant N:O/Wilga group (PVYN:O or PVYN-Wilga) [3–5]. PVYNTN is characterized by its ability to induce potato tuber necrotic ringspot disease (PTNRD) in sensitive potato cultivars [5–7], whereas PVYN:O is defined by its reaction to PVYO-specific antibody (i.e., PVYO-serotype) but causing veinal necrosis on tobacco plants (i.e., PVYN pathotype) [5, 8]. Two types of PVYNTN, one recombinant and the other non-recombinant, have been identified [4, 7, 9]. The former is represented by PVYNTN-Hun  and has been referred to as European (Eu)-PVYNTN[7, 11–13], and the latter is represented by PVYNTN-Tu 660  and has been referred to as North American (NA)-PVYNTN[7, 11, 12, 14]. Both Eu-PVYNTN and NA-PVYNTN react to PVYN-specific antibody [11, 13]. Recently, a new recombinant PVYNTN variant type has been identified in Syria [15, 16] and China . The variant type that includes the isolates PVYNTN-NW and PVYNTN-HN2  reacts to PVYO-antibody and induces veinal necrosis on tobacco and PTNRD on sensitive potato cultivars [9, 16]. Reverse transcription-PCR (RT-PCR) based genotyping has been successfully used to characterize the genome features of the Eu-PVYNTN-like isolate PVYNTN-HN1 and the PVYNTN-NW-like isolate PVYNTN-HN2 in China . Here we report the differential responses of Physalis floridana to PVYNTN-HN1 and PVYNTN-HN2 infections. PVYNTN-HN1 and PVYN induced mottling on P. floridana whereas PVYNTN-HN2, PVYO and PVYN:O induced severe symptoms including leaf and stem necrosis, leaf-drop and stunting. The results, together with the genome make-ups of various PVY isolates, suggest that the CP gene plays a significant role in symptom induction in P. floridana, consistent with the results reported by Bukovinszki et al..
The greenhouse maintained PVY isolates PVYNTN-HN1 (formerly PVY sample 1 ), PVYNTN-HN2, PVYN-Jg, PVYO-RB and PVYN:O-Mb58 in 'Russet Burbank' plants/tubers [5, 7, 9, 11–13] were used in this study. PVYNTN-HN1 and PVYNTN-HN2 were obtained in China , while the rest were from Canada [5, 7, 11–13]. All isolates have been characterized molecularly by P1 gene- and recombinant joint (RJ)-based RT-PCR assays [12, 13], pathologically by tobacco- and potato-based bioassays, and serologically by PVYO- and PVYN-antibody-based ELISA assays [5, 7, 9, 11–13, 18]. Moreover, except for PVYNTN-HN1, all of the isolates have been sequenced fully (PVYNTN-HN-2, PVYN-Jg, PVYO-RB) or partially (PVYN:O-Mb58) (accession numbers are HM367076, AY166867, GQ200836, AY745493 for PVYO-RB, PVYN-Jg, PVYNTN-HN-2, and PVYN:O-Mb58 respectively). To better understand the isolate PVYNTN-HN1, especially to reveal the exact nucleotide locations of the recombinant joints that had been detected by RT-PCR , the complete genome of PVYNTN-HN1 was sequenced. The same nine sets of PCR primers (for primer sequences, see reference ) that had been used to clone/sequence various isolates of PVY [5, 7, 9, 18] were used. Each primer pair resulted in a DNA fragment of 1.0 to 1.3 kb, overlapping with adjacent fragments with approximately 100 bp at each end. Each fragment was cloned into a pGM-T cloning vector (TIANGEN Biotech, Beijing, China) according to the manufacturer's instructions; and two clones of each fragment were sequenced from both forward and reverse directions using the universal T7 promoter and SP6-promoter primers at the Sangon Biological Engineering Technology & Services Co. Ltd (Shanghai, China). The complete genome sequence (GenBank accession number HQ631374) was confirmed by re-sequencing overlapping cDNA clones obtained from a separate experiment from RNA isolated from PVYNTN-HN1 infected tobacco leaves. Sequence identities were analyzed using BLAST (http://www.ncbi.nlm.nih.gov/BLAST). For detection of the recombinant events, complete nucleotide sequences of various PVY isolates were aligned using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html) . The aligned sequences served as inputs for similarity scanning using the program SimPlot [20, generously provided by the author at http://sray.med.som.jhmi.edu/]. The resulting similarities were plotted along the nucleotide sequences of the virus genome.
Identities between isolate PVYNTN-HN1 and other isolates of Potato virus Y (PVY) at both nucleic acid and protein levels
Sequence Identity (%) (Nucleic acid, Protein)
Gene nucleotide, size (bp)
Protein size (aa)
It has been known that PVYO induces necrosis in Physalis floridana, whereas PVYN incites mottling in this species . Using N/O hybrids comprised of the chimeric genome of PVYN-N605  and PVYO, the symptom formation on P. floridana due to PVY infection was mapped to the CP gene region . Because of the varied genome compositions among the isolates (Figure 1D), they could be used to investigate the putative role of genome segment(s) of PVY in symptom development on P. floridana, as done on tobacco [5, 24]. Severe symptoms including leaf and stem necrosis, leaf-drop and stunting were observed on P. floridana plants infected with PVYO-RB, PVYN:O-Mb58 and PVYNTN-HN2 three weeks after inoculation (Figure 1C), and as time progressed, the symptoms became more distinct. The isolate PVYN:O-Mb58 led to plant death five weeks after the inoculation. On the other hand, mild symptoms, mainly mottling, were observed on PVYN-Jg and PVYNTN-HN1 infected P. floridana plants (Figure 1C). Taken together, it can be concluded that the CP gene originated from PVYO is likely responsible for the severe symptoms in PVYO-, PVYN:O- or PVYNTN-HN2-infected P. floridana plants. These results, together with the results obtained using artificial PVY chimeras , demonstrate that the CP gene, especially the 5' proximal segment (nt 8572-9183) of the gene, plays a critical role in symptom formation in P. floridana upon PVY infection, and determines the pathoginicity of PVY isolates. The 3' proximal segment of NIb gene (nt 8136-8570) does not appear to be involved in the symptom formation in P. floridana as suggested by Bukovinszki et al.. It is also noteworthy that the different symptoms incited by different PVY types/isolates in tobacco, potato and P. floridana can be used to uncover the genome compositions of the virus.
The research was supported by the Ministry of Science and Technology of China under the project # 20073346 to XX and CH and by Agriculture and Agri-Food Canada (AAFC) under the projects #50 and #1389 to XN. XH was a receipt of the Ministry of Education of China-AAFC Ph.D. Student Internship Program (2007-2008); XN is an adjunct professor at Hunan Agricultural University.
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