Development of near homozygous lines for diploid hybrid TPS breeding in potatoes.

Diploid inbred-based F1 hybrid True Potato Seed (DHTPS) breeding is a novel technique to transform potato breeding and cultivation across the globe. Significant efforts are being made to identify elite diploids, dihaploids and develop diploid inbred lines for heterosis exploitation in potatoes. Self-incompatibility is the first obstacle for developing inbred lines in diploid potatoes, which necessitates the introgression of a dominant S locus inhibitor gene (Sli) for switching self-incompatibility to self-compatibility. We evaluated a set of 357 diploid clones in different selfing generations for self-compatibility and degree of homozygosity using Kompetitive Allele Specific PCR (KASP) Single Nucleotide Polymorphism (SNP) markers. A subset of 10 KASP markers of the Sli candidate region on chromosome 12 showed an association with the phenotype for self-compatibility. The results revealed that the selected 10 KASP markers for the Sli gene genotype could be deployed for high throughput rapid screening of self-compatibility in diploid populations and to identify new sources of self-compatibility. The homozygosity assessed through 99 KASP markers distributed across all the chromosomes of the potato genome was 20-78 % in founder diploid clones, while different selfing generations, i.e., S0, S1, S2 and S3 observed 36.1-80.4, 56.9-82.8, 59.5-85.4 and 73.7-87.8 % average homozygosity, respectively. The diploid plants with ∼80 % homozygosity were also observed in the first selfing generation, which inferred that homozygosity assessment in the early generations itself could identify the best plants with high homozygosity to speed up the generation of diploid inbred lines.

Kaolinite nanoclay-shielded dsRNA drenching for management of Globodera pallida: An environmentally friendly pest management approach.

Globodera pallida, an obligate sedentary endoparasite, is a major economic pest that causes substantial potato yield losses. This research aimed to study the effects of gene silencing of three FMRFamide-like peptides (FLPs) genes to reduce G. pallida infestation on potato plants by using kaolinite nanoclay as a carrier to deliver dsRNAs via drenching. A dsRNA dosage of 2.0 mg/ml silenced flp-32c by 89.5%, flp-32p by 94.6%, and flp-2 by 94.3%. J2s incubated for 5 and 10 h showed no phenotypic changes. However, J2s of G. pallida efficiently uptake dsRNA of all targeted genes after 15 h of incubation. On the other hand, J2s that had been kept for 24 h had a rigid and straight appearance. Under fluorescence microscopy, all dsRNA-treated nematodes showed fluorescein isothiocyanate (FITC) signals in the mouth, nervous system, and digestive system. The untreated population of J2s did not show any FITC signals and was mobile as usual. The drenching of potato cultivar Kufri Jyoti with the dsRNA-kaolinite formulations induced deformation and premature death of J2s, compared with untreated J2s that entered J3 or J4 stages. This study validates that the nanocarrier-delivered RNAi system could be employed effectively to manage G. pallida infestations.

Tandem Expression of a Mobile RNA and Its RNA-Binding Protein(s) Enhances Tuber Productivity in Potato.

A significant number of discoveries in past two decades have established the importance of long-distance signaling in controlling plant growth, development, and biotic and abiotic stress responses. Numerous mobile signals, such as mRNAs, proteins, including RNA-binding proteins, small RNAs, sugars, and phytohormones, are shown to regulate various agronomic traits such as flowering, fruit, seed development, and tuberization. Potato is a classic model tuber crop, and several mobile signals are known to govern tuber development. However, it is unknown if these mobile signals have any synergistic effects on potato crop improvement. Here, we employed a simple innovative strategy to test the cumulative effects of a key mobile RNA, StBEL5, and its RNA-binding proteins, StPTB1, and -6 on tuber productivity of two potato cultivars, Solanum tuberosum cv. Désirée and subspecies andigena, using a multi-gene stacking approach. In this approach, the coding sequences of StBEL5 and StPTB1/6 are driven by their respective native promoters to efficiently achieve targeted expression in phloem for monitoring tuber productivity. We demonstrate that this strategy resulted in earliness for tuberization and enhanced tuber productivity by 2-4 folds under growth chamber, greenhouse, and field conditions. This multi-gene stacking approach could be adopted to other crops, whose agronomic traits are governed by mobile macromolecules, expanding the possibilities to develop crops with improved traits and enhanced yields.

Identification of genes governing resistance to PCN (Globodera rostochiensis) through transcriptome analysis in Solanum tuberosum.

Potato cyst nematodes (PCNs) are major pests worldwide that affect potato production. The molecular changes happening in the roots upon PCN infection are still unknown. Identification of transcripts and genes governing PCN resistance will help in the development of resistant varieties. Hence, differential gene expression of compatible (Kufri Jyoti) and incompatible (JEX/A-267) potato genotypes was studied before (0 DAI) and after (10 DAI) inoculation of Globodera rostochiensis J2s through RNA sequencing (RNA-Seq). Total sequencing reads generated ranged between 33 and 37 million per sample, with a read mapping of 48-84% to the potato reference genome. In the infected roots of the resistant genotype JEX/A-267, 516 genes were downregulated, and 566 were upregulated. In comparison, in the susceptible genotype Kufri Jyoti, 316 and 554 genes were downregulated and upregulated, respectively. Genes encoding cell wall proteins, zinc finger protein, WRKY transcription factors, MYB transcription factors, disease resistance proteins, and pathogenesis-related proteins were found to be majorly involved in the incompatible reaction after PCN infection in the resistant genotype, JEX/A-267. Furthermore, RNA-Seq results were validated through quantitative real-time PCR (qRT-PCR), and it was observed that ATP, FLAVO, CYTO, and GP genes were upregulated at 5 DAI, which was subsequently downregulated at 10 DAI. The genes encoding ATP, FLAVO, LBR, and GP were present in > 1.5 fold before infection in JEX-A/267 and upregulated 7.9- to 27.6-fold after 5 DAI; subsequently, most of these genes were downregulated to 0.9- to 2.8-fold, except LBR, which was again upregulated to 44.4-fold at 10 DAI.

RNA-seq analysis reveals an early defense response to tomato leaf curl New Delhi virus in potato cultivar Kufri Bahar.

Potatoes in India are very susceptible to apical leaf curl disease, which causes severe symptoms and greater yield losses. Because the majority of potato cultivars are susceptible to the virus, it is crucial to discover sources of resistance and investigate the mechanism of resistance/susceptibility in potato cultivars. In this study, the gene expression profile of two potato cultivars, Kufri Bahar (resistant) and Kufri Pukhraj (susceptible), varying in their level of resistance to ToLCNDV, was analyzed using RNA-Seq. The Ion ProtonTM system was used to sequence eight RiboMinus RNA libraries from inoculated and uninoculated potato plants at 15 and 20 days after inoculation (DAI). The findings indicated that the majority of differentially expressed genes (DEGs) were cultivar-or time-specific. These DEGs included genes for proteins that interact with viruses, genes linked with the cell cycle, genes for proteins involved in defense, transcription and translation initiation factors, and plant hormone signaling pathway genes. Interestingly, defense responses were generated early in Kufri Bahar, at 15 DAI, which may have impeded the replication and spread of ToLCNDV. This research provides a genome-wide transcriptional analysis of two potato cultivars with variable levels of ToLCNDV resistance. At an early stage, we observed suppression of genes that interact with viral proteins, induction of genes associated with restriction of cell division, genes encoding defense proteins, AP2/ERF transcription factors, and altered expression of zinc finger protein genes, HSPs, JA, and SA pathway-related genes. Our findings add to a greater comprehension of the molecular basis of potato resistance to ToLCNDV and may aid in the development of more effective disease management techniques.

CRISPR/Cas9-mediated editing of phytoene desaturase (PDS) gene in an important staple crop, potato.

The gene editing using the CRISPR/Cas9 system has become an important biotechnological tool for studying gene function and improving crops. In this study, we have used CRISPR/Cas9 system for editing the phytoene desaturase gene (PDS) in popular Indian potato cultivar Kufri Chipsona-I. A construct (pHSE401) carrying two target gRNAs with glycine tRNA processing system under the control of Arabidopsis U6 promoter and the Cas9 protein was constructed and transformed in potato plants using Agrobacterium-mediated genetic transformations. The regeneration efficiency of 45% was observed in regenerated plants, out of which 81% of the putative transformants shoot lines exhibited mutant or bleached phenotype (albinism). The deletion mutations were detected within the StPDS gene in the genotyped plants and a mutation efficiency of 72% for gRNA1 and gRNA2 has been detected using Sanger sequencing. Hence, we set up a CRISPR/Cas9-mediated genome editing protocol which is efficient and generates mutations (deletions) within StPDS gene in potato. The bleached phenotype is easily detectable after only few weeks after Agrobacterium-mediated transformation. This is the first report as a proof of concept for CRISPR/Cas9-based editing of PDS gene in Indian potato cv. Kufri Chipsona-I. This study demonstrates that CRISPR/Cas9 can be used to edit genes at high frequency within the genome of the potato for various traits. Therefore, this study will aid in creating important mutants for modifying molecular mechanisms controlling traits of agronomic importance.

Generation of Asynaptic Mutants in Potato by Disrupting StDMC1 Gene Using RNA Interference Approach.

Fixing the genomic composition and multiplication through true potato seed (TPS) is an important challenge in autotetraploid potato. Disrupted meiotic cDNA (DMC1) is a meiotic gene that plays a central role in DNA recombination through crossing over in meiosis. Using the Arabidopsis DMC1 (AtDMC1) gene sequence, we retrieved Solanum tuberosum DMC1(StDMC1) from the diploid potato genome, and subsequently, sense and antisense regions of the StDMC1 gene were amplified in potato cv. Kufri Jyoti. The sense and antisense fragments were confirmed by Sanger-sequencing and cloned in the pRI101 vector. Agrobacterium-mediated transformation of the RNAi construct resulted in 44% transformation efficiency, and a total of 137 mutant lines were obtained. These mutant lines were further validated through pollen viability testing, and selected lines were used for gene expression analysis. The acetocarmine-based pollen staining showed reduced pollen viability ranging from 14 to 21% in four DMC1 mutant lines (DMC4-37, DMC4-41, DMC6-20, and DMC6-21), as compared to the Kufri Jyoti control plants, which on average exhibited 78% pollen viability. The phenotypic data was supported by the reduced expression of the StDMC1 gene in these four mutant lines compared to the control Kufri Jyoti. The results confirmed the generation of StDMC1 knockdown lines. This is the first report of StDMC1 mutant line generation in tetraploid potatoes and will be a step forward in generating non-recombinant mutants through sexual reproduction in potatoes.

Genotypic variations for tuber nutrient content, dry matter and agronomic traits in tetraploid potato germplasm.

Nutrient deficiencies lead to various health issues and are common worldwide. Potato germplasm is a rich source of natural variations and genetic variability present in it can be exploited for developing nutrient-rich high-yielding potato varieties. In this study, variations in the yield, dry matter (DM) and mineral nutrients concentrations were evaluated in both peeled and unpeeled tubers of 243 highly diverse tetraploid potato accessions. These were raised under field conditions for two consecutive years. The germplasm studied has a wider range of variations in peeled tubers DM (13.71-27.80%), Fe (17.08-71.03 mg/kg), Zn (9.55-34.78 mg/kg), Cu (2.13-13.25 mg/kg), Mn (7.04-25.15), Ca (117.4-922.5 mg/kg), Mg (656.6-1510.6 mg/kg), S (1121.3-3765.8 mg/kg), K (1.20-3.09%), P (0.21-0.50%) and Mo (53.6-1164.0 ppb) concentrations compared to popular Indian potato varieties. Higher nutrient concentrations in whole tubers compared to tuber flesh suggest that these are present in high concentration in the tuber peripheral layers and peeling off the tubers results in the loss of nutrients. Highest loss due to peeling off the tubers was observed in Fe (35.63%) followed by Cu (22.80%), Mn (21.69%), Ca (21.27%), Mg (12.89%), K (12.75%), Zn (10.13%), and Mo (9.87%). The GCV and PCV for all the traits in peeled tubers ranged from 9.67 to 29.91%, and 13.84 to 43.32%, respectively. Several significant positive correlations were observed among the parameters and the first two principal components accounted for 39.37% of total variations. The results of this study will pave a way for the development of nutrient-rich high-yielding potato varieties. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01197-1.

Establishment of a one-step reverse transcription recombinase polymerase amplification assay for the detection of potato virus S.

Potato virus S (PVS) is a noteworthy threat to the propagation of healthy seed potatoes. Accurate and speedy detection is critical for effective PVS management. In the present study, an isothermal-based one-step reverse transcription-recombinase polymerase amplification (RT-RPA) approach was developed to detect PVS infection in potato leaves and tubers. A primer set based on the coat protein gene successfully amplified a 158 bp product out of three primer sets examined. The amplification reaction took less than 30 min to complete with no account of cross-reactivity with major potato viruses. Additionally, amplification of RT-RPA products was performed on the heating system and/or water bath at 38-42 °C. The results of sensitivity analysis revealed that one-step RT-RPA has shown 100 times higher sensitivity than routine RT-PCR for the detection of PVS in infected leaves. Furthermore, ten times higher sensitivity of RT-RPA was observed in infected tubers. The methodology was simplified further by the use of template RNA extracted using a cellular disc paper-based extraction method that detected the PVS more effectively than purified total RNA. PVS was detected in 175 samples (leaves and tubers each) of several potato varieties using this innovative technique. To our acquaintance, this is the first report of one-step RT-RPA using a basic RNA extract derived through cellular disc paper that is significantly sensitive and precise for PVS detection in potatoes. The advantages of one-step RT-RPA in terms of proficiency, robustness, and the availability of a highly pure RNA template make it an attractive choice for seed accreditation, resistance breeding, and field inspections.

A new record of Asia II 5 genetic group of Bemisia tabaci (Gennadius) in the major potato growing areas of India and its relationship with tomato leaf curl New Delhi virus infecting potato.

The whitefly, Bemisia tabaci (Gennadius), is responsible for significant yield losses in many crops, including potato, by sucking the phloem sap and transmitting a number of plant viruses. B. tabaci is a complex of cryptic species which is commonly designated as genetic groups. The B. tabaci genetic groups differ biologically with respect to host plant preference, insecticidal resistance, reproduction capacity, and ability to transmit begomoviruses. Therefore, understanding genetic variation among populations is important for establishing crop-specific distribution profile and management. We sequenced the mitochondrial cytochrome oxidase I (mtCOI) gene of B. tabaci collected from major potato growing areas of India. BLAST analysis of the 24 mtCOI sequences with reference Gene Bank sequences revealed four B. tabaci genetic groups prevailing in this region. mtCOI analysis exhibited the presence of Asia II 1, Asia II 5, Asia 1, and MEAM1 B. tabaci genetic groups. Our study highlighted that a new genetic group Asia II 5 has been detected in Indo-Gangetic Plains. Further virus-vector relationship study of ToLCNDV with Asia II 5 B. tabaci revealed that females are efficient vector of this virus as compared to males. This behavior of females might be due to their ability to acquire more virus titer than males. This study will help in better understanding of whitefly genetic group mediated virus diseases.

Rapid and sensitive detection of potato virus X by one-step reverse transcription-recombinase polymerase amplification method in potato leaves and dormant tubers.

Potato virus X (PVX), is a serious threat to global potato production. A simple and rapid detection method is imperative for PVX diagnosis and early management. In this study, an isothermal one-step reverse transcription-recombinase polymerase amplification (RT-RPA) method was optimized for the quick and convenient detection of PVX in potato leaves and tubers. Our results revealed that this one-step RT-RPA method was highly efficient than the conventional reverse transcription-polymerase chain reaction (RT-PCR). The amplification reaction was free from cross-reactivity with other common potato viruses and completed within 30 min. Moreover, this RT-RPA assay did not require a thermocycler based specific temperature phase amplification and can be easily performed using a simple heating block or water bath at a temperature range of 39-42 °C. The sensitivity assay demonstrated that the developed one-step RT-RPA method was 100 times more sensitive than a routine one-step RT-PCR. Initially, the purified total RNA as the template isolated from infected leaves of potato was used for the detection of PVX. One-step RT-RPA was later performed using cellular disc paper-based simple RNA extract as a template that could detect the virus more efficiently than purified total RNA. The performance of the one-step RT-RPA assay was further evaluated using 500 field samples of leaves and tubers representing different cultivars and geographical regions. To our knowledge, this is the first report of rapid, sensitive, and reliable detection of PVX infection by one-step RT-RPA using cellular disc paper-based simple RNA extract from leaves and dormant tubers of potato. It is superior to the common RT-PCR assay in terms of its versatility, quickness, and independence of highly purified RNA template and can be adopted as a substitute to RT-PCR as an effective technique for seed potato certification, quarantine, breeding, and field surveys.

Molecular characterization and infectivity analysis of tomato leaf curl New Delhi virus isolates infecting potato.

Nucleotide sequence of complete genome of a new isolate (KAN-6) of tomato leaf curl New Delhi virus (ToLCNDV) from Kanpur, Uttar Pradesh, India was determined. Sequence analysis indicated that it shared maximum identity to ToLCNDV isolates from pumpkin and ashgourd. Infectious clones of isolate KAN-6 along with two other ToLCNDV isolates (MOD-21 & FAI-19) obtained from potato fields of Modipuram and Faizabad, India were produced and used in symptom expression studies in N. benthamiana and potato plants through agro-inoculation. These isolates produced different symptoms both in N. benthamiana and potato. Severe symptoms of yellow mottling, downward curling and stunted growth were observed in N. benthamiana plants inoculated with KAN-6. MOD-21-inoculated plants also showed downward curling, stunted growth, but yellow mottling was observed only in older leaves whereas FAI-19-inoculated plants produced only downward curling symptoms. In case of potato, typical symptoms of apical leaf curl disease were observed in cultivar Kufri Pukhraj inoculated with MOD-21 and KAN-6 that are similar to those produced by virus-infected plants in the field. However, MOD-21 produced more prominent yellow mosaic symptoms as compared to KAN-6. FAI-19 produced only restricted yellow spots in Kufri Pukhraj. Only mild symptoms appeared in KAN-6 and no symptoms were observed in MOD-21- and FAI-19-inoculated Kufri Bahar plants which is known to show lowest seed degeneration under field conditions. Analysis of genomic components indicated that these isolates had 94.8-94.9% and 87.9-97.3% identity among them in DNA A and DNA B, respectively. The results of the study indicate the association of ToLCNDV isolates of different symptomatology with apical leaf curl disease of potato. This is also a first experimental demonstration of Koch's postulate for a begomovirus associated with apical leaf curl disease of potato.Author names: Please confirm if the author names (Swarup Kumar Chakrabarti) are presented accurately and in the correct sequence (given name, middle name/initial, family name).Yes. It is correct. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02752-5.

Comparison of various RNA extraction methods, cDNA preparation and isolation of calmodulin gene from a highly melanized isolate of apple leaf blotch fungus Marssonina coronaria.

Marssonina coronaria causes apple blotch disease resulting in severe premature defoliation, and is distributed in many leading apple-growing areas in the world. Effective, reliable and high-quality RNA extraction is an indispensable procedure in any molecular biology study. No method currently exists for RNA extraction from M. coronaria that produces a high quantity of melanin-free RNA. Therefore, we evaluated eight RNA extraction methods including manual and commercial kits, to yield a sufficient quantity of high-quality and melanin-free RNA. Manual methods used here resulted in low quality and black colored RNA pellets showing the presence of melanin, despite all the modifications employed to original procedures. However, these methods when coupled with clean up resulted in melanin-free RNA. On the other hand, all commercial kits used were able to yield high-quality melanin-free RNA having variable yields. TRIzol™ Reagent + RNA Clean & Concentrator™-5 and Ambion-PureLink® RNA Mini Kit were found to be the best methods as the RNA extracted with these methods from 15 day old fungal culture grown on solid medium were free of melanin with good yield. RNA extracted by this improved methodology was applied for RT-PCR, subsequent PCR amplification, and isolation of calmodulin gene sequences from M. coronaria and infected apple leaf pieces. These methods are more time effective than traditional methods and take only an hour to complete. To our knowledge, this is the first report on the method of isolation of high-quality RNA for cDNA synthesis as well as isolation of the calmodulin gene sequence from this fungus.

MiRNA160 is associated with local defense and systemic acquired resistance against Phytophthora infestans infection in potato.

To combat pathogen infection, plants employ local defenses in infected sites and elicit systemic acquired resistance (SAR) in distant tissues. MicroRNAs have been shown to play a significant role in local defense, but their association with SAR is unknown. In addition, no such studies of the interaction between potato and Phytophthora infestans have been reported. We investigated the role of miR160 in local and SAR responses to P. infestans infection in potato. Expression analysis revealed induced levels of miR160 in both local and systemic leaves of infected wild-type plants. miR160 overexpression and knockdown plants exhibited increased susceptibility to infection, suggesting that miR160 levels equivalent to those of wild-type plants may be necessary for mounting local defense responses. Additionally, miR160 knockdown lines failed to elicit SAR, and grafting assays indicated that miR160 is required in both local and systemic leaves to trigger SAR. Consistently, SAR-associated signals and genes were dysregulated in miR160 knockdown lines. Furthermore, analysis of the expression of defense and auxin pathway genes and direct regulation of StGH3.6, a mediator of salicylic acid-auxin cross-talk, by the miR160 target StARF10 revealed the involvement of miR160 in antagonistic cross-talk between salicylic acid-mediated defense and auxin-mediated growth pathways. Overall, our study demonstrates that miR160 plays a crucial role in local defense and SAR responses during the interaction between potato and P. infestans.

Key players associated with tuberization in potato: potential candidates for genetic engineering.

Tuberization in potato (Solanum tuberosum L.) is a complex biological phenomenon which is affected by several environmental cues, genetic factors and plant nutrition. Understanding the regulation of tuber induction is essential to devise strategies to improve tuber yield and quality. It is well established that short-day photoperiods promote tuberization, whereas long days and high-temperatures inhibit or delay tuberization. Worldwide research on this complex biological process has yielded information on the important bio-molecules (proteins, RNAs, plant growth regulators) associated with the tuberization process in potato. Key proteins involved in the regulation of tuberization include StSP6A, POTH1, StBEL5, StPHYB, StCONSTANS, Sucrose transporter StSUT4, StSP5G, etc. Biomolecules that become transported from "source to sink" have also been suggested to be important signaling candidates regulating the tuberization process in potatos. Four molecules, namely StSP6A protein, StBEL5 RNA, miR172 and GAs, have been found to be the main candidates acting as mobile signals for tuberization. These biomolecules can be manipulated (overexpressed/inhibited) for improving the tuberization in commercial varieties/cultivars of potato. In this review, information about the genes/proteins and their mechanism of action associated with the tuberization process is discussed.

An insight into differentially regulated genes in resistant and susceptible genotypes of potato in response to tomato leaf curl New Delhi virus-[potato] infection.

Apical leaf curl disease, caused by tomato leaf curl New Delhi virus-[potato] (ToLCNDV-[potato]) is one of the most important viral diseases of potato in India. Genetic resistance source for ToLCNDV in potato is not identified so far. However, the cultivar Kufri Bahar is known to show lowest seed degeneration even under high vector levels. Hence, microarray analysis was performed to identify differentially regulated genes during ToLCNDV-[potato] infection in a resistant (Kufri Bahar) and a susceptible cultivar (Kufri Pukhraj). Under artificial inoculation conditions, in Kufri Pukhraj, symptom expressions started at 15days after inoculation (DAI) and then progressed to severe symptoms, whereas no or only very mild symptoms were observed in Kufri Bahar up to 35 DAI. Correspondingly, qPCR assay indicated a high viral load in Kufri Pukhraj and a very low viral load in Kufri Bahar. Microarray analysis showed that a total of 1111 genes and 2588 genes were differentially regulated (|log2 (Fold Change)|>2) in Kufri Bahar and Kufri Pukhraj, respectively, following ToLCNDV-[potato] infection. Gene ontology and mapman analyses revealed that these altered transcripts were involved in various biological & metabolic processes. Several genes with unknown functions were 5 to 100 fold expressed after virus infection and further experiments are necessary to ascertain their role in disease resistance or susceptibility. This study gives an insight into differentially regulated genes in response to ToLCNDV-[potato] infection in resistant and susceptible cultivars and could serve as the basis for the development of new strategies for disease management.

Regulation, overexpression, and target gene identification of Potato Homeobox 15 (POTH15) - a class-I KNOX gene in potato.

Potato Homeobox 15 (POTH15) is a KNOX-I (Knotted1-like homeobox) family gene in potato that is orthologous to Shoot Meristemless (STM) in Arabidopsis. Despite numerous reports on KNOX genes from different species, studies in potato are limited. Here, we describe photoperiodic regulation of POTH15, its overexpression phenotype, and identification of its potential targets in potato (Solanum tuberosum ssp. andigena). qRT-PCR analysis showed a higher abundance of POTH15 mRNA in shoot tips and stolons under tuber-inducing short-day conditions. POTH15 promoter activity was detected in apical and axillary meristems, stolon tips, tuber eyes, and meristems of tuber sprouts, indicating its role in meristem maintenance and leaf development. POTH15 overexpression altered multiple morphological traits including leaf and stem development, leaflet number, and number of nodes and branches. In particular, the rachis of the leaf was completely reduced and leaves appeared as a bouquet of leaflets. Comparative transcriptomic analysis of 35S::GUS and two POTH15 overexpression lines identified more than 6000 differentially expressed genes, including 2014 common genes between the two overexpression lines. Functional analysis of these genes revealed their involvement in responses to hormones, biotic/abiotic stresses, transcription regulation, and signal transduction. qRT-PCR of selected candidate target genes validated their differential expression in both overexpression lines. Out of 200 randomly chosen POTH15 targets, 173 were found to have at least one tandem TGAC core motif, characteristic of KNOX interaction, within 3.0kb in the upstream sequence of the transcription start site. Overall, this study provides insights to the role of POTH15 in controlling diverse developmental processes in potato.

Host-mediated gene silencing of a single effector gene from the potato pathogen Phytophthora infestans imparts partial resistance to late blight disease.

RNA interference (RNAi) has proved a powerful genetic tool for silencing genes in plants. Host-induced gene silencing of pathogen genes has provided a gene knockout strategy for a wide range of biotechnological applications. The RXLR effector Avr3a gene is largely responsible for virulence of oomycete plant pathogen Phytophthora infestans. In this study, we attempted to silence the Avr3a gene of P. infestans through RNAi technology. The P. infestans inoculation resulted in lower disease progression and a reduction in pathogen load, as demonstrated by disease scoring and quantification of pathogen biomass in terms of Pi08 repetitive elements, respectively. Transgenic plants induced moderate silencing of Avr3a, and the presence and/or expression of small interfering RNAs, as determined through Northern hybridization, indicated siRNA targeted against Avr3a conferred moderate resistance to P. infestans. The single effector gene did not provide complete resistance against P. infestans. Although the Avr3a effector gene could confer moderate resistance, for complete resistance, the cumulative effect of effector genes in addition to Avr3a needs to be considered. In this study, we demonstrated that host-induced RNAi is an effective strategy for functional genomics in oomycetes.