Molecular insights on potato yellow vein crinivirus infections in the highlands of Colombia.

Potato yellow vein virus (PYVV) was detected in potatoes grown in the Central highlands, north of Bogotá (~3000 m altitude), Colombia. At this altitude viral whitefly vectors are largely absent, but infection persists because of the use of uncertified tubers. Plants with typical PYVV-induced yellowing symptoms, as well as with atypical yellowing or non-symptomatic symptoms were sampled at three separate geographical locations. PYVV presence was assessed by RT-PCR, and several plants were subjected to high-throughput sequencing (HTS) of their small RNA (sRNA) populations. Complete or almost complete sequences of four PYVV isolates were thus reconstructed, all from symptomatic plants. Three viral isolates infected plants singly, while the fourth co-infected the plant together with a potyvirus. Relative proportions of sRNAs to each of the three crinivirus genomic RNAs were found to remain comparable among the four infections. Genomic regions were identified as hotspots of sRNA formation, or as regions that poorly induced sRNAs. Furthermore, PYVV titres in the mixed versus single infections remained comparable, indicating an absence of synergistic/antagonistic effects of the potyvirus on the accumulation of PYVV. Daughter plants raised in the greenhouse from tubers of the infected, field-sampled plants displayed mild PYVV infection symptoms that disappeared with time, demonstrating the occurrence of recovery and asymptomatic infection phenotypes in this pathosystem.

Validation of molecular response of tuberization in response to elevated temperature by using a transient Virus Induced Gene Silencing (VIGS) in potato.

Temperature plays an important role in potato tuberization. The ideal night temperature for tuber formation is ~17 °C while temperature beyond 22 °C drastically reduces the tuber yield. Moreover, high temperature has several undesirable effects on the plant and tubers. Investigation of the genes involved in tuberization under heat stress can be helpful in the generation of heat-tolerant potato varieties. Five genes, including StSSH2 (succinic semialdehyde reductase isoform 2), StWTF (WRKY transcription factor), StUGT (UDP-glucosyltransferase), StBHP (Bel1 homeotic protein), and StFLTP (FLOWERING LOCUS T protein), involved in tuberization and heat stress in potato were investigated. The results of our microarray analysis suggested that these genes regulate and function as transcriptional factors, hormonal signaling, cellular homeostasis, and mobile tuberization signals under elevated temperature in contrasting KS (Kufri Surya) and KCM (Kufri Chandramukhi) potato cultivars. However, no detailed report is available which establishes functions of these genes in tuberization under heat stress. Thus, the present study was designed to validate the functions of these genes in tuber signaling and heat tolerance using virus-induced gene silencing (VIGS). Results indicated that VIGS transformed plants had a consequential reduction in StSSH2, StWTF, StUGT, StBHP, and StFLTP transcripts compared to the control plants. Phenotypic observations suggest an increase in plant senescence, reductions to both number and size of tubers, and a decrease in plant dry matter compared to the control plants. We also establish the potency of VIGS as a high-throughput technique for functional validation of genes.

Fast, Precise, and Reliable Multiplex Detection of Potato Viruses by Loop-Mediated Isothermal Amplification.

Potato is an important staple food crop in both developed and developing countries. However, potato plants are susceptible to several economically important viruses that reduce yields by up to 50% and affect tuber quality. One of the major threats is corky ringspot, which is a tuber necrosis caused by tobacco rattle virus (TRV). The appearance of corky ringspot symptoms on tubers prior to commercialization results in ≈ 45% of the tubers being downgraded in quality and value, while ≈ 55% are declared unsaleable. To improve current disease management practices, we have developed simple diagnostic methods for the reliable detection of TRV without RNA purification, involving minimalized sample handling (mini), subsequent improved colorimetric loop-mediated isothermal amplification (LAMP), and final verification by lateral-flow dipstick (LFD) analysis. Having optimized the mini-LAMP-LFD approach for the sensitive and specific detection of TRV, we confirmed the reliability and robustness of this approach by the simultaneous detection of TRV and other harmful viruses in duplex LAMP reactions. Therefore, our new approach offers breeders, producers, and farmers an inexpensive and efficient new platform for disease management in potato breeding and cultivation.

Transmission modes affect the population structure of potato virus Y in potato.

Transmission is a crucial part of a viral life cycle and transmission mode can have an important impact on virus biology. It was demonstrated that transmission mode can influence the virulence and evolution of a virus; however, few empirical data are available to describe the direct underlying changes in virus population structure dynamics within the host. Potato virus Y (PVY) is an RNA virus and one of the most damaging pathogens of potato. It comprises several genetically variable strains that are transmitted between plants via different transmission modes. To investigate how transmission modes affect the within-plant viral population structure, we have used a deep sequencing approach to examine the changes in the genetic structure of populations (in leaves and tubers) of three PVY strains after successive passages by horizontal (aphid and mechanical) and vertical (via tubers) transmission modes. Nucleotide diversities of viral populations were significantly influenced by transmission modes; lineages transmitted by aphids were the least diverse, whereas lineages transmitted by tubers were the most diverse. Differences in nucleotide diversities of viral populations between leaves and tubers were transmission mode-dependent, with higher diversities in tubers than in leaves for aphid and mechanically transmitted lineages. Furthermore, aphid and tuber transmissions were shown to impose stronger genetic bottlenecks than mechanical transmission. To better understand the structure of virus populations within the host, transmission mode, movement of the virus within the host, and the number of replication cycles after transmission event need to be considered. Collectively, our results suggest a significant impact of virus transmission modes on the within-plant diversity of virus populations and provide quantitative fundamental data for understanding how transmission can shape virus diversity in the natural ecosystems, where different transmission modes are expected to affect virus population structure and consequently its evolution.

Planting a Problem: Examining the Spread of Seed-Borne Potato Virus Y.

Potato virus Y (PVY) is among the most economically impactful potato pathogens, yet the spread of PVY from infected seed potatoes within commercial potato fields has not been adequately studied. Test lots containing various seed-borne PVY levels were created by mixing different proportions of seed pieces from healthy and infected tubers drawn from the same seed source. These seed lots were planted in commercial potato fields near the Teton Seed Potato Management Area from 2010 to 2012. Regression analyses on data from these test plots produced models of the in-season spread of PVY originating from infected seed. Conventional ordinary least squares techniques were supplemented with the use of quantile regression; the resulting models indicate the significance of seed-borne PVY on end-of-season infection levels and highlight the need of seed potato buyers to review postharvest testing results.

Optimization of an isothermal recombinase polymerase amplification method for real-time detection of Potato virus Y O and N types in potato.

Potato virus Y (PVY) is a global challenge for potato production and the leading cause of seed crop downgrading and rejection for certification. Accurate and timely diagnosis is key to effective control of PVY. Here we optimized the isothermal recombinase polymerase amplification (RPA) for accurate detection of different PVY O and N types that were tested, present in different tissues of potato plants including tubers with a primer set that specifically targets the highly conserved pipo region within the viral genome. Combined with a simplified preparation of the template by tissue homogenization, we established a rapid RPA procedure, which can allow real time detection in less than 10 min with a fluorescent probe. Specificity of the reaction was determined by the lack of cross-reactivity with other common potato viruses. Although RPA reagents remain more expensive than PCR reagents, RPA technology is equivalent in that results can be visualized by gel electrophoresis or with a fluorescent probe with greater sensitivity; and it is superior to the common PCR-based assay in its versatility, speed, and lack of need for a highly purified RNA template.

Alterations of the viroid regions that interact with the host defense genes attenuate viroid infection in host plant.

Understanding in intimate details how the viroid interaction with host's defense genes is a cornerstone for developing viroid resistant plants. In this present study, small RNAs (sRNA) derived from Potato spindle tuber viroid (PSTVd) were studied in silico in order to detect any interactions with the serine threonine kinase receptor, a transmembrane protein that plays a role in disease resistance in plants. Using molecular biology techniques, it was determined that PSTVd infection negatively affects at least three serine threonine kinase receptors as well as with three other genes that are known to be involved in the overall development of the tomato plants. The transient expression of these putative PSTVd-sRNAs, using the microRNA sequence as a backbone, in tomato plants induced phenotypes similar to viroid infection. Mutants created by altering the sequence of PSTVd in these regions failed to infect the tomato plant. The data presented here illustrates the importance of these regions in viroid survival, and suggests a possible avenue of exploration for the development of viroid resistant plants.

Chromogenic detection of yam mosaic virus by closed-tube reverse transcription loop-mediated isothermal amplification (CT-RT-LAMP).

A closed-tube reverse transcription loop-mediated isothermal amplification (CT-RT-LAMP) assay was developed for the detection of yam mosaic virus (YMV, genus Potyvirus) infecting yam (Dioscorea spp.). The assay uses a set of six oligonucleotide primers targeting the YMV coat protein region, and the amplification products in YMV-positive samples are visualized by chromogenic detection with SYBR Green I dye. The CT-RT-LAMP assay detected YMV in leaf and tuber tissues of infected plants. The assay is 100 times more sensitive in detecting YMV than standard RT-PCR, while maintaining the same specificity.

Molecular characterization of variants of a new 'rule-breaking' tobacco rattle virus RNA2 in potatoes.

In potato tubers showing pronounced corky ringspot symptoms, two related 'rule-breaking" tobacco rattle virus (TRV) RNA2s, named Da-2 and Db-2, were identified. Their coat protein (cp) genes are preceded on the 5' side by an additional gene for a 35 kDa protein for which no relationships with previously described TRV genes or their expression products were found. With estimated 4296 and 4247 nucleotides (nts) the two RNAs are the longest TRV RNA2s described so far. The difference in size between Da-2 and Db-2 is due to a duplication of a stretch of 49 nts in the 5' untranslated region of Da-2. An alignment of TRV coat proteins (cp) revealed that up to about amino acid (aa) 176 they form two rather uniform groups. The much shorter C-terminal parts of the cps, however, differ considerably in size and composition. With 56 aa this C-terminal part is much longer in the Da and Db sequences than in all other TRV cps. It differs in 18 positions in the two strains whereas their N-terminal 184 aa differ only in two positions. - In young potato plants developing from bud-cuttings of TRV Db-infected potato tubers which had been planted in soil free of virus and nematodes a gradual degradation of Db-2 was observed. In the newly formed rootlets already five days after planting a deletion of 80 nts was observed in the putative 2b gene which in other TRV strains encodes a protein necessary for nematode transmission. Thirty three days after planting the entire 2b gene, 119 nts at the 3'end of the cp gene and a portion of the original 3'untranslated region of Db-2 had been lost in the newly formed roots, leaves and stolons. The gene for the 35kDa protein was the only one which was not affected by deletions which seems to emphasize its importance for the virus. Fifty days after planting only TRV RNA1, but no TRV RNA2 were detectable.

Assessing Potato Cultivar Sensitivity to Tuber Necrosis Caused by Potato mop-top virus.

Potato mop-top virus (PMTV) causes mop top disease in potato. This disease can result in a decline in tuber quality causing economic losses to growers due to the production of necrotic lesions and discolored tissue in infected tubers. Due to the soilborne nature of PMTV, identifying and developing host resistance against the virus is considered the best disease management option. Very little is known about the sensitivity of U.S. potato cultivars to PMTV-induced tuber necrosis. The current study is aimed at investigating the sensitivity of a large number of potato cultivars to PMTV-induced tuber necrosis. Sixty-three cultivars representing all market-types were evaluated in North Dakota over a 2-year period for virus-induced tuber necrosis incidence and severity. PMTV-induced tuber necrosis (P < 0.0001) and severity (P < 0.0001) were significantly different among cultivars. Cultivars were categorized into sensitive, insensitive, and moderately sensitive/insensitive groups based on the virus-tuber induced necrosis data from both years. Based on data from ND trials, six cultivars (Red Endeavor, Viking, Dakota Jewel, Dark Red Norland, Nicolet, and Modoc) were rated as sensitive and 43 were rated as insensitive to PMTV-induced tuber necrosis. Four cultivars, including Bannock Russet, Gemstar Russet, Lelah, and Waneta showed zero PMTV incidence over 2 years. These results will help growers in making individual or coordinated decisions for the management of PMTV-induced tuber necrosis under field and storage conditions.

Assessing Potato Cultivar Sensitivity to Tuber Necrosis Caused by Tobacco rattle virus.

Tobacco rattle virus (TRV) causes the economically important corky ring spot disease in potato. Chemical control is difficult due to the soilborne nature of the TRV-transmitting nematode vector, and identifying natural host resistance against TRV is considered to be the optimal control measure. The present study investigated the sensitivity of 63 cultivars representing all market types (evaluated at North Dakota and Washington over 2 years) for the incidence of TRV-induced tuber necrosis and severity. This article also investigates the cultivar-location interaction (using a mixed-effects model) for TRV-induced necrosis. TRV-induced tuber necrosis (P < 0.0001) and severity (P < 0.0001) were significantly different among cultivars evaluated separately in North Dakota and Washington trials. Mixed-effects model results of pooled data (North Dakota and Washington) demonstrated that the interaction of cultivar and location had a significant effect (P = 0.03) on TRV-induced necrosis. Based on the virus-induced tuber necrosis data from both years and locations, cultivars were categorized into sensitive, moderately sensitive, insensitive, and moderately insensitive groups. Based on data from North Dakota, 10 cultivars, including Bintje, Centennial Russet, Ciklamen, Gala, Lelah, Oneida Gold, POR06V12-3, Rio Colorado, Russian Banana, and Superior, were rated as insensitive to TRV-induced tuber necrosis. Similar trials assessing TRV sensitivity among cultivars conducted in Washington resulted in a number of differences in sensitivity rankings compared with North Dakota trials. A substantial shift in sensitivity of some potato cultivars to TRV-induced tuber necrosis was observed between the two locations. Four cultivars (Centennial Russet, Oneida Gold, Russian Banana, and Superior) ranked as insensitive for North Dakota trials were ranked as sensitive for Washington trials. These results can assist the potato industry in making cultivar choices to reduce the economic impact of TRV-induced tuber necrosis.

Processing of Potato Spindle Tuber Viroid RNAs in Yeast, a Nonconventional Host.

Potato spindle tuber viroid (PSTVd) is a circular, single-stranded, noncoding RNA plant pathogen that is a useful model for studying the processing of noncoding RNA in eukaryotes. Infective PSTVd circles are replicated via an asymmetric rolling circle mechanism to form linear multimeric RNAs. An endonuclease cleaves these into monomers, and a ligase seals these into mature circles. All eukaryotes may have such enzymes for processing noncoding RNA. As a test, we investigated the processing of three PSTVd RNA constructs in the yeast Saccharomyces cerevisiae Of these, only one form, a construct that adopts a previously described tetraloop-containing conformation (TL), produces circles. TL has 16 nucleotides of the 3' end duplicated at the 5' end and a 3' end produced by self-cleavage of a delta ribozyme. The other two constructs, an exact monomer flanked by ribozymes and a trihelix-forming RNA with requisite 5' and 3' duplications, do not produce circles. The TL circles contain nonnative nucleotides resulting from the 3' end created by the ribozyme and the 5' end created from an endolytic cleavage by yeast at a site distinct from where potato enzymes cut these RNAs. RNAs from all three transcripts are cleaved in places not on path for circle formation, likely representing RNA decay. We propose that these constructs fold into distinct RNA structures that interact differently with host cell RNA metabolism enzymes, resulting in various susceptibilities to degradation versus processing. We conclude that PSTVd RNA is opportunistic and may use different processing pathways in different hosts.IMPORTANCE In higher eukaryotes, the majority of transcribed RNAs do not encode proteins. These noncoding RNAs are responsible for mRNA regulation, control of the expression of regulatory microRNAs, sensing of changes in the environment by use of riboswitches (RNAs that change shape in response to environmental signals), catalysis, and more roles that are still being uncovered. Some of these functions may be remnants from the RNA world and, as such, would be part of the evolutionary past of all forms of modern life. Viroids are noncoding RNAs that can cause disease in plants. Since they encode no proteins, they depend on their own RNA and on host proteins for replication and pathogenicity. It is likely that viroids hijack critical host RNA pathways for processing the host's own noncoding RNA. These pathways are still unknown. Elucidating these pathways should reveal new biological functions of noncoding RNA.

The viability of lytic bacteriophage ΦD5 in potato-associated environments and its effect on Dickeya solani in potato (Solanum tuberosum L.) plants.

Dickeya solani is one of the most important pectinolytic phytopathogens responsible for high losses in potato, especially in seed potato production in Europe. Lytic bacteriophages can affect the structure of the host population and may influence spread, survival and virulence of the pathogen and in consequence, infection of the plant. In this study, we aimed to acquire information on the viability of the broad host lytic bacteriophage ΦD5 on potato, as well as to apprehend the specific effect of this bacteriophage on its host D. solani type-strain in different settings, as a preliminary step to target co-adaptation of phages and host bacteria in plant environment. Viability of the ΦD5 phage in tuber extract, on tuber surface, in potting compost, in rainwater and on the leaf surface, as well as the effect of copper sulfate, were examined under laboratory conditions. Also, the interaction of ΦD5 with the target host D. solani in vitro and in compost-grown potato plants was evaluated. ΦD5 remained infectious in potato tuber extract and rain water for up to 72 h but was inactivated in solutions containing 50 mM of copper. The phage population was stable for up to 28 days on potato tuber surface and in potting compost. In both, tissue culture and compost-grown potato plants, ΦD5 reduced infection by D. solani by more than 50%. The implications of these findings are discussed.

Molecular and Biochemical Examination of Spraing Disease in Potato Tuber in Response to Tobacco rattle virus Infection.

Field-grown tubers of potato were examined for infection by Tobacco rattle virus (TRV) and consequent production of corky ringspot or spraing symptoms. A microarray study identified genes that are differentially expressed in tuber tissue in response to TRV infection and to spraing production, suggesting that hypersensitive response (HR) pathways are activated in spraing-symptomatic tubers. This was confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) of a selected group of HR-related genes and by histochemical staining of excised tuber tissue with spraing symptoms. qRT-PCR of TRV in different regions of the same tuber slice showed that nonsymptomatic areas contained higher levels of virus relative to spraing-symptomatic areas. This suggests that spraing formation is associated with an active plant defense that reduces the level of virus in the infected tuber. Expression of two of the same plant defense genes was similarly upregulated in tubers that were infected with Potato mop-top virus, a virus that also induces spraing formation.

Insight on Genes Affecting Tuber Development in Potato upon Potato spindle tuber viroid (PSTVd) Infection.

Potato (Solanum tuberosum L) is a natural host of Potato spindle tuber viroid (PSTVd) which can cause characteristic symptoms on developing plants including stunting phenotype and distortion of leaves and tubers. PSTVd is the type species of the family Pospiviroidae, and can replicate in the nucleus and move systemically throughout the plant. It is not well understood how the viroid can affect host genes for successful invasion and which genes show altered expression levels upon infection. Our primary focus in this study is the identification of genes which can affect tuber formation since viroid infection can strongly influence tuber development and especially tuber shape. In this study, we used a large-scale method to identify differentially expressed genes in potato. We have identified defence, stress and sugar metabolism related genes having altered expression levels upon infection. Additionally, hormone pathway related genes showed significant up- or down-regulation. DWARF1/DIMINUTO, Gibberellin 7-oxidase and BEL5 transcripts were identified and validated showing differential expression in viroid infected tissues. Our study suggests that gibberellin and brassinosteroid pathways have a possible role in tuber development upon PSTVd infection.

Small RNA Derived from the Virulence Modulating Region of the Potato spindle tuber viroid Silences callose synthase Genes of Tomato Plants.

The tomato (Solanum lycopersicum) callose synthase genes CalS11-like and CalS12-like encode proteins that are essential for the formation of callose, a major component of pollen mother cell walls; these enzymes also function in callose formation during pathogen infection. This article describes the targeting of these callose synthase mRNAs by a small RNA derived from the virulence modulating region of two Potato spindle tuber viroid variants. More specifically, viroid infection of tomato plants resulted in the suppression of the target mRNAs up to 1.5-fold, depending on the viroid variant used and the gene targeted. The targeting of these mRNAs by RNA silencing was validated by artificial microRNA experiments in a transient expression system and by RNA ligase-mediated rapid amplification of cDNA ends. Viroid mutants incapable of targeting callose synthase mRNAs failed to induce typical infection phenotypes, whereas a chimeric viroid obtained by swapping the virulence modulating regions of a mild and a severe variant of Potato spindle tuber viroid greatly affected the accumulation of viroids and the severity of disease symptoms. These data provide evidence of the silencing of multiple genes by a single small RNA derived from a viroid.

The amino acid 419 in HC-Pro is involved in the ability of PVY isolate N605 to induce necrotic symptoms on potato tubers.

The ability to induce the potato tuber necrosis ringspot disease (PTNRD) is a property shared by PVY isolates belonging to different groups (e.g. PVY(N) and PVY(O)) and variants (e.g. PVY(NTN) and PVY(N)-W). The identification of viral molecular determinant(s) involved in the expression of PTNRD symptoms is essential for (i) an easier detection of tuber necrosis isolates and (ii) an improvement of our knowledge on the epidemiology of this potato disease. A reverse genetic approach associated with a biological typing of a collection of PVY chimeras and mutants indicated that residue E419 of the HC-Pro protein is linked to the ability of PVY to induce tuber necrosis on four PTNRD-susceptible potato cultivars. Indeed, the substitution of the N-type glutamic acid (E) in O-type aspartic acid (D) at position 419 in the HC-Pro cistron prevents the expression of tuber necrosis on infected tubers without reducing the virulence of the corresponding E/D419 mutant. This result opens opportunities for the future studies on potato/PVY interactions.

Molecular and serological methods for the diagnosis of viruses in potato tubers.

Viruses cause important diseases to potato crops. Monitoring virus content in plant material for quarantine or seed certification scheme purposes is essential to prevent the spread of viruses and to minimize the impact of viral diseases. There are currently two main methods for virus diagnosis in potato tubers: growing-on ELISA testing which requires breaking tuber dormancy followed by an ELISA test on grown plantlets and direct real-time RT-PCR testing on tubers. This chapter will describe both methods that can be adapted for large-scale virus testing activities.

Immunocapture-Multiplex RT-PCR for the Simultaneous Detection and Identification of Plant Viruses and Their Strains: Study Case, Potato Virus Y (PVY).

Immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) is a sensitive, reproducible, and robust method for the detection and identification of RNA viruses. The IC step provides a simple method to isolate virus particles from plant tissue, particularly when inhibitory substances are present, and thus enables subsequent use of RT-PCR amplification for large-scale virus testing and typing. The multiplex format of the PCR is often used for the detection and identification of multiple virus/strain simultaneously to save time, labor, and cost. Potato virus Y (PVY) is one of the most economically important viruses infecting potato worldwide. PVY exists as a complex of at least nine strains and many more unclassified recombinants that vary in their genome structures, phenotypes, and their economic importance. In the current chapter, a detailed protocol of an IC-based, multiplex RT-PCR assay for the detection and identification of various PVY strains is described.

Detection of Potato spindle tuber viroid and Other Related Viroids by a DIG Labelled RNA Probe.

Viroids can cause diseases of considerable economic importance; in Europe the main concern is with pospiviroids that may affect the tomato and potato industries. Methods for detection are required that are both sensitive and robust. The detection method described here is a probe hybridization method with a commercially available digoxigenin (DIG) labelled full-length Potato spindle tuber viroid (PSTVd) RNA probe. This method detects PSTVd and all other known pospiviroids.