Each treatment inoculation set consisted of five plants and was replicated three times three blocks. The secondary or tuberborne infection showed a similar trend. The number of PVY-positive plants emerging from tubers harvested from plants inoculated at different time points declined starting from W4 and dropped to zero for W7. When the primary and secondary infections for W4 to W8 were compared in , the upper leaves of 10 plants were found negative for PVY no systemic infection but produced at least one PVY-infected tuber.
These included four plants of W4, four plants of W5, one plant of W6, and one plant of W8. In the case of W6 and W8, only a single tuber per plant was found PVY infected in a subsequent grow out. In , only four plants were found negative for the primary PVY infection no systemic infection but produced PVY-positive tubers identified during the secondary infection testing. Of these four plants, two of the W4-infected plants produced PVY-infected tubers both of the two tubers tested per plant , and the other two came from two separate W6-infected plants, producing one PVY-infected tuber of the two tubers tested per plant.
However, one plant each from W6- and W8-infected plants was found to be PVY positive for the primary infection, but all of their tubers produced PVY-negative plants. Table 1. Numbers of systemically infected potato plants that emerged from tubers originating from plants inoculated with potato virus Y PVY NTN at 8 different consecutive weeks after transplanting and two seasons of greenhouse experiments in and Plants of W1 and W2 inoculations had average tuber counts of 2.
No significant differences in tuber counts per plant were recorded for W3 to W8 inoculations compared with the healthy control Fig. The effects of the inoculation with the recombinant strain of potato virus Y PVY , PVY NTN , at 8 consecutive weeks after transplanting W1 to W8; x axis on the average of tuber counts per cultivar Yukon Gold plant y axis in greenhouse experiments in and Total numbers of tubers were recorded at harvest time for each plant and used in the analysis.
Values with the same lower case letters are not significantly different. A pooled analysis of both years, and , indicated significant differences for the tuber yield based on the plant age at inoculation. The yield for W1 and W2 inoculations was significantly lower than that of the W3 inoculation, which was significantly lower than that of the W4 inoculation. W4 tuber yield was lower than that from W5 to W8. No significant differences were recorded at W5 to W8 compared with the healthy control Fig.
The effects of the inoculation with the recombinant strain of potato virus Y PVY , PVY NTN , at 8 consecutive weeks after transplanting W1 to W8; x axis on the average of tuber yield per cultivar Yukon Gold plant y axis in greenhouse experiments in and plotted separately. A pooled analysis of and experiments showing the effects of the inoculation with the recombinant strain of potato virus Y PVY , PVY NTN , at 8 consecutive weeks after transplanting W1 to W8; x axis on the average of yield per plant grams; y axis of cultivar Yukon Gold plants in greenhouse experiments.
Symptoms of potato tuber necrotic ringspot disease. A, Various severity levels of tuber necrosis associated with inoculation at different weeks after transplanting. B, Example of harvested tubers from plants inoculated at 8 consecutive weeks after transplanting in the greenhouse experiment of Table 2.
The percentage of tubers of the potato cultivar Yukon Gold displaying tuber necrotic ringspots at 1 month postharvest from plants inoculated with potato virus Y at 8 different consecutive weeks after transplanting and two seasons of greenhouse experiments in and Tubers from plants inoculated at W1 and W2 showed the most severe symptoms, with more than six necrotic rings per tuber; this number decreased significantly for plants inoculated at W3 to about necrotic 2.
Tubers from plants inoculated at W5 and W6 had a very low average number of necrotic arcs or dark halos around the stolon end, which was not significantly different from W7, W8, and the healthy control Fig. The PTNRD severity was scaled on each tuber by counting the numbers of necrotic rings and arcs at 1 month postharvest. ARR in potato is a type of field resistance that was recognized and studied in Europe starting in the s Beemster ; Gibson ; Sigvald From the W5 inoculation onward, the systemic movement of the virus was almost completely blocked, with very few plants found infected systemically Fig.
This impairment of the systemic movement in the inoculated plants starting at the W4 inoculation generally correlated with the reduction of the secondary infection starting from W4 inoculation-derived tubers, which meant also an impairment in the translocation of the virus into tubers for plants inoculated starting at the W4 inoculation.
However, later in the season W4 to W8 , a few plants tested negative for PVY in the upper noninoculated leaves no systemic movement of the virus ; nevertheless, they produced a small number of PVY-infected tubers, particularly at W4 to W6 inoculations. The same finding was reported by Jones and Vincent : PVY was detected in progeny tubers without being detected in the foliage of primarily inoculated plants showing local HR.
Consistently, there were rare cases where late PVY infection may not lead to systemic spread of the virus in the foliage yet may result in production of PVY-infected tubers. Conversely, some upper noninoculated leaves of the plants inoculated later in the season W4 to W8 inoculations were found to be positive for PVY systemically infected with PVY but produced PVY-negative tubers impairment of virus translocation to tubers.
On several occasions, 9 PVY-positive or PVY-negative plants of plants at the W4 to W8 inoculation time point for both and produced a mixture of infected and healthy tubers. The per plant averages of tuber counts and yield were not affected by the late inoculation W5 to W8 Figs.
Indeed, the tuber counts almost doubled in plants inoculated at W4 to W8 and equaled the healthy control. Systemic PVY NTN infection in plants of the W3 inoculation did not reduce the tuber counts significantly compared with the healthy control but resulted in significantly smaller tubers and lower average yield Figs.
In , an increase of about g in the average yield was recorded for plants inoculated at W4 to W8 and the healthy control compared with the same treatments of the experiment. Inoculated leaves exhibited HR regardless of the plant age, and the virus was readily detected in those inoculated leaves but not in the upper noninoculated leaves of plants from late inoculations W5 to W8. Previous studies, however, demonstrated ARR in cultivar-strain combinations where no specific N gene was known to be expressed.
In the case of another potyvirus, bean common mosaic necrosis virus BCMNV , two types of the resistance in common bean were found uncoupled the HR type expressed by the I gene and the recessive resistance restricting systemic movement of BCMNV and expressed by the bc-1 gene Feng et al. From a practical standpoint, the ARR may have great promise in the control of PVY for both seed and commercial potato production Beemster ; Gibson ; Sigvald Consequently, if applicable to other cultivar-strain combinations and in the absence of a massive, early-season infestation with PVY, ARR provides sufficient protection to the crop, and commercial potato producers may be assured that the damage to the potato yields and tuber quality caused by the late season PVY infection should be negligible as long as they use high-quality certified seed potato.
For seed potato producers, however, this study points at the early season as a critical timeframe for the efficient protection of potato crops from PVY infection. If such control early in the season is provided, it would greatly reduce the number of infection sources in the field available for aphid transmission later in the season and reduce the overall PVY translocation into progeny tubers in otherwise asymptomatic plants missed during visual summer inspections.
We thank J. Chojnacky for laboratory and greenhouse help and J. Durrin for providing potato plantlets. Home Plant Disease Vol. Lisa T. William J. Alexander V. Tran 1 William J. Price 2 Alexander V. Add to favorites Download Citations Track Citations. View article. View as image HTML. Literature Cited Asama, K. Agr Exp Stn Google Scholar Baldauf, P.
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Strain-specific hypersensitive and extreme resistance phenotypes elicited by Potato virus Y among 39 potato cultivars released in three world regions over a year period. Continuous and emerging challenges of Potato virus Y in potato. Therefore, developing novel approaches to control plant viruses is crucial to meet the demands of a growing world population.
Once genome replication and assembly of virion particles is completed inside the host plant, mature virions or sometimes naked viral genomes spread cell-to-cell through plasmodesmata by interacting with the virus-encoded movement protein MP. Khufri Ashoka. Overall, our findings provide a robust technology to generate PLRV-resistant potato plants, which can be extended to other species. Moreover, this approach also contributes to the study of genome translocation mechanisms of plant viruses.
Plant viruses cause serious damage to crops, and it is estimated that economic loss caused by viral pathogens ranks the second compared to that caused by other pathogens 1. These viruses infect potato plants most of the times in combination with other pathogens or sometimes individually.
Since potato is vegetatively propagated, the chances of virus dissemination through progeny tubers are high. Tubers used for planting in the subsequent season can harbour latent viruses, which adversely affect plant growth and yield 3.
Assembly of virion particles is a critical step during viral maturation process. In most plant viruses, assembly of virion particles occurs through the nucleation of capsid proteins CPs around nucleic acids 4. The movement protein MP helps in either the passage of virion particles through plasmodesmata PD enlargement or direct transfer of genomic material in the form of nucleoprotein complex to the adjacent cells 5.
The mature plant virion particles spread to long distances by passing from one cell to another. They eventually infect a new host through vectors such as insects, nematodes, mites, and fungi. Targeting virus assembly or maturation process can be an effective strategy to develop PLRV resistance in plants.
One of the most successful approaches to build plant resistance has been to introduce a part of the viral genomic sequence into the plant either to induce RNA silencing against viral RNA or to express intact or modified viral proteins or RNAs that disturb the viral infection cycle 6 , 7 , 8. RNA interference RNAi technology has emerged as a potential tool to target virus assembly for developing resistant crops 6.
RNA silencing, a conserved regulatory mechanism of gene expression in eukaryotes, is triggered by dsRNA-provoking gene silencing through sequence-specific degradation of complementary mRNA transcripts post-transcriptional gene silencing 9.
Systemic infection of plant cells by viruses requires several crucial steps. After replication, plant virus particles spread to non-infected adjacent cells as virions or viral ribonucleoprotein vRNP complexes by cell-to-cell. The cell-to-cell movement of viruses through PD requires virus-encoded MPs MPs help in the enlargement of PD pore size and active transport of the viral nucleic acid into the adjacent cell, thereby allowing local spread of viruses in plants As MP plays a crucial role in PLRV transmission, we sought to analyse its amino acid sequence to identify conserved motifs and use them to develop a potential method of developing virus-resistant plants.
First, we analysed the amino acid sequence of MP using bioinformatics tools. Our extensive bioinformatic analysis revealed four putative motifs, namely walker A, B, sensor, and arginine finger I and II, which possess ATP-binding and hydrolysis activities and are spread throughout the polypeptide chain of MP. We hypothesized that these motifs have a direct role in the active transportation of virions or nucleic acids through PD. To validate this hypothesis, we cloned, expressed, and purified MP in Escherichia coli.
Further, we ascertained its function using nucleotide-stimulated ATP hydrolysis activity. As MP homologues are present in most plant viruses, their inhibition via RNAi can be a potential method for generating virus-resistant plants. Multiple sequence alignments were generated using ClustalW 15 and were manually corrected for domain superimpositions.
The digested fragment was ligated to the pET28b plasmid, which was linearized with the same restriction enzymes. The ligated DNA was transformed into E. The accuracy of the cloned DNA inserts was further confirmed by sequencing. Finally, the MP recombinant plasmids were transformed into E. The supernatant containing the protein of interest was loaded onto pre-equilibrated Ni—NTA beads ThermoFisher Scientific and the His-tagged protein was eluted using mM imidazole.
Colorimetric detection of inorganic phosphate Pi was performed using a malachite green-based method The malachite green reagent 0. In this method, Pi reacts with molybdate and forms a green-coloured complex, which can be quantified at nm.
Two hundred microliter of the malachite green reagent was added to the reaction mixture, and Pi generated through ATPase activity was quantified at nm. The complexes were visualized in UV light. The transformed cells of A. For agroinfiltration, A. The cells were then incubated at room temperature for 2—3 h before agroinfiltration. Khufri Ashoka plants were agroinfiltrated with 2 mL of syringe directly into the phloem and leaf as well.
Whole plants were covered with a transparent plastic bag for 3—4 days. All experiments were repeated thrice with five plants in each experiment for each siRNA constructs and control. PLRV infected tubers were grown in a controlled environment. Ten days post agroinfiltration, newly tertiary leaves were harvested for RNAi analysis.
The gels were stained for 10 min with 0. The membrane was then blocked for 15 min with blocking buffer using gentle shaking at room temperature followed by incubation with hybridization buffer containing stabilized streptavidin-HRP conjugate for an additional 15 min. Tertiary leaves of agroinfiltrated plants and control leaves positive and negative of potato 0.
The substrate p-nitrophenyl phosphate was added to the plate for developing and absorbance at nm was measured. The absorbances of samples and controls were normalized to that of the positive control.
MPs are known as plant virus-encoded factors that interact with PD to mediate the intercellular spread of viruses in planta. All the putative motifs necessary for ATP binding and hydrolysis are present in the middle domain of the viral MP, except Walker B orange and arginine finger II red which are situated at N and C-terminus respectively Fig.
Further, we observed that the Arginine finger I pink and II motifs red , which are located 9—10 residues downstream of Walker B motif and 18 residues downstream of Walker A, and the sensor motif green present about two residues further downstream of the Arginine finger I motif are strictly conserved across the strains of PLRV analysed in this study Fig.
Multiple sequence alignments, generated using ClustalW, were manually corrected for domain superimpositions. The number s in brackets represent the number of amino acids. Schematics are drawn approximately to the scale and represent the approximate consensus of representative homologs.
Lane 1: Purified protein and lane 2: Marker. Negative controls with only buffer were also taken into account while performing the reactions. With increasing protein concentrations, the rate of ATP hydrolysis increased Fig. The nucleotide binding evidence of recombinant MP was further confirmed by gel shift assay Fig.
Specific primers were designed for the amplification of the gene sequence of MP in sense and antisense orientation. PCR amplification of an oligo dT-primed first-strand cDNA template prepared from the mRNA extracted from infected leaves yielded bp bands of both sense and antisense orientation Fig.
Further, the orientation of genes was confirmed through sequencing IDT. A Amplified cDNA fragments were analyzed by electrophoresis on 0. These findings indicated that siRNA constructs hampered or suppressed viral translocation and multiplication in the plants. Higher expression of siRNA in the leaves at 10 dpi lane 1 was observed as compared to 5 dpi lane 2 , whereas empty vector mock agro-infected leaves did not show any siRNA lane 3.
Amplified cDNA fragments bp were analyzed by electrophoresis on 0. D Actin PCR was performed as an internal control. The agro-infected regions of the leaf were used to isolate total RNA which was analysed for the accumulation of siRNAs 5 and 10 days after inoculation. The siRNAs were detected in the agro-infected region from day 5, 10 Fig. The bp amplified DNA fragment corresponding to the CP gene of PLRV was detected in the tertiary leaves of plants agroinfiltrated with the empty vector and control leaves; however, it was not detected in the tertiary leaves of plants agroinfiltrated with the MP siRNA constructs.
The absorbance values of all the samples were normalized to that of the positive control. The mean absorbance values for all the samples are presented in Fig. We noted that the relative absorbance values of the samples from plants containing the siRNA constructs were two times that of the negative controls. A Absorbance values of all the samples were normalized to that of the positive control.
B Hypothetical model for the transportation of mature virion particles pathway I; lower panel and their genomes through plasmodesmata with the assistance of ATP hydrolysis by MP pathway II; upper panel.
Plant viruses cannot exploit membrane fusion-based routes of entry as described for animal viruses. Once plant viruses enter the cell by means of a vector, they need to enhance the restricted pore size of PD connecting channels between plant cells. Thus, the transportation of virion particles through PD from one cell to another cell is a critical step during infection of plant viruses Fig. Previous studies have revealed that PD allow the cell-to-cell trafficking of plant viruses with the help of viral encoded MPs 20 , 21 , In addition, MPs are also involved in PD gating that allows the direct intercellular movement of viral genomes Fig.
Various host factors such as cytoskeleton, endoplasmic reticulum, and other endomembrane interact with MPs to regulate complex mechanisms of PD gating Although several studies have reported the detailed mechanism of the movement of virions or vRNP through PD 19 , 23 , 24 , 25 , the active role of MP in this movement remains unclear. Our sequence analysis Fig. The crucial Arginine finger II motif is distantly placed in the atomic model from the Walker A, B, sensor, and arginine finger I motifs, which form a cleft or active site for the binding of ATP.
Ranjan, Unpublished data. In the future, we plan to replace the critical amino acid residues of these putative ATPase motifs through site directed mutagenesis, which will further dissect the function of these motifs, especially in terms of active genome translocation through PD. Since the ATPase activity is believed to act as driving force for viral genome transportation, the ability of the purified proteins to hydrolyse ATP was analysed Fig. The agroinfiltrated plants did not show PLRV infection.
Suppression of viral infection could be attributed to the reduced expression of MP due to its silencing by the siRNA constructs. The present study indicated that the transient expression of MP constructs resulted in specific and efficient inhibition of PLRV. These findings further confirm the earlier reports and suggest that MP has a direct role in the active cell-to-cell transportation of the viral genome and virions and is thus involved in systemic translocation The inhibition of the expression of MP by gene silencing is an efficient and promising method to introduce resistance to PLRV As PLRV causes severe crop yield losses in the potato growing regions worldwide, our findings will be helpful for developing PLRV-resistant potato crops.
This approach can also be applied to an extensive range of plant species against various viral diseases. Antiviral strategies in plants based on RNA silencing. Acta , — Google Scholar. Kreuze, J. Viral diseases in potato. Chapter Google Scholar. Kumar, R. Plant Pathol. Chelikani, V. Genome segregation and packaging machinery in Acanthamoeba polyphaga Mimivirus is reminiscent of bacterial apparatus. Sunpapao, A. Virus-induced symptoms in plants: A review of interactions between viral trafficking and RNA silencing.
Philipp Agric. Khalid, A. Small RNA based genetic engineering for plant viral resistance: Application in crop protection. Article Google Scholar. Goldbach, R. Resistance mechanisms to plant viruses: An overview. Virus Res. Lodge, J. Broad-spectrum virus resistance in transgenic plants expressing pokeweed antiviral protein.
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